Methods and compositions for cns delivery of heparan n-sulfatase

ABSTRACT

The present invention provides, among other things, compositions and methods for CNS delivery of lysosomal enzymes for effective treatment of lysosomal storage diseases. In some embodiments, the present invention includes a stable formulation for direct CNS intrathecal administration comprising a heparan N-sulfatase (HNS) protein, salt, and a polysorbate surfactant for the treatment of Sanfilippo Syndrome Type A.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/077,046, filed Mar. 22, 2016, which is a divisional applicationof issued U.S. Pat. No. 9,320,711, filed Jun. 25, 2011 which claimspriority to U.S. Provisional Patent Application Ser. Nos. 61/358,857filed Jun. 25, 2010; 61/360,786, filed Jul. 1, 2010; 61/387,862, filedSep. 29, 2010; 61/435,710, filed Jan. 24, 2011; 61/442,115, filed Feb.11, 2011; 61/476,210, filed Apr. 15, 2011; and 61/495,268 filed on Jun.9, 2011; the entirety of each of which is hereby incorporated byreference. This application relates to US applications entitled “CNSDelivery of Therapeutic Agents;” filed on even date; “Methods andCompositions for CNS Delivery of Iduronate-2-Sulfatase,” filed on evendate; “Methods and Compositions for CNS Delivery ofβ-Galactocerebrosidase,” filed on even date; “Methods and Compositionsfor CNS Delivery of Arylsulfatase A,” filed on even date; “Treatment ofSanfilippo Syndrome Type B,” filed on even date; the entirety of each ofwhich is hereby incorporated by reference.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listing(submitted electronically as a .txt file named 200688-1336_SL.txt onJul. 16, 2018). The .txt file was generated on Jul. 16, 2018 and is9,135 bytes in size. The entire contents of the Sequence Listing areherein incorporated by reference.

BACKGROUND

Enzyme replacement therapy (ERT) involves the systemic administration ofnatural or recombinantly-derived proteins and/or enzymes to a subject.Approved therapies are typically administered to subjects intravenouslyand are generally effective in treating the somatic symptoms of theunderlying enzyme deficiency. As a result of the limited distribution ofthe intravenously administered protein and/or enzyme into the cells andtissues of the central nervous system (CNS), the treatment of diseaseshaving a CNS etiology has been especially challenging because theintravenously administered proteins and/or enzymes do not adequatelycross the blood-brain barrier (BBB).

The blood-brain barrier (BBB) is a structural system comprised ofendothelial cells that functions to protect the central nervous system(CNS) from deleterious substances in the blood stream, such as bacteria,macromolecules (e.g., proteins) and other hydrophilic molecules, bylimiting the diffusion of such substances across the BBB and into theunderlying cerebrospinal fluid (CSF) and CNS.

There are several ways of circumventing the BBB to enhance braindelivery of a therapeutic agent including direct intra-cranialinjection, transient permeabilization of the BBB, and modification ofthe active agent to alter tissue distribution. Direct injection of atherapeutic agent into brain tissue bypasses the vasculature completely,but suffers primarily from the risk of complications (infection, tissuedamage, immune responsive) incurred by intra-cranial injections and poordiffusion of the active agent from the site of administration. To date,direct administration of proteins into the brain substance has notachieved significant therapeutic effect due to diffusion barriers andthe limited volume of therapeutic that can be administered.Convection-assisted diffusion has been studied via catheters placed inthe brain parenchyma using slow, long-term infusions (Bobo, et al.,Proc. Natl. Acad. Sci. U.S.A 91, 2076-2080 (1994); Nguyen, et al. J.Neurosurg. 98, 584-590 (2003)), but no approved therapies currently usethis approach for long-term therapy. In addition, the placement ofintracerebral catheters is very invasive and less desirable as aclinical alternative.

Intrathecal (IT) injection, or the administration of proteins to thecerebrospinal fluid (CSF), has also been attempted but has not yetyielded therapeutic success. A major challenge in this treatment hasbeen the tendency of the active agent to bind the ependymal lining ofthe ventricle very tightly which prevented subsequent diffusion.Currently, there are no approved products for the treatment of braingenetic disease by administration directly to the CSF.

In fact, many believed that the barrier to diffusion at the brain'ssurface, as well as the lack of effective and convenient deliverymethods, were too great an obstacle to achieve adequate therapeuticeffect in the brain for any disease.

Many lysosomal storage disorders affect the nervous system and thusdemonstrate unique challenges in treating these diseases withtraditional therapies. There is often a large build-up ofglycosaminoglycans (GAGs) in neurons and meninges of affectedindividuals, leading to various forms of CNS symptoms. To date, no CNSsymptoms resulting from a lysosomal disorder has successfully beentreated by any means available.

Thus, there remains a great need to effectively deliver therapeuticagents to the brain. More particularly, there is a great need for moreeffective delivery of active agents to the central nervous system forthe treatment of lysosomal storage disorders.

SUMMARY OF THE INVENTION

The present invention provides an effective and less invasive approachfor direct delivery of therapeutic agents to the central nervous system(CNS). The present invention is, in part, based on unexpected discoverythat a replacement enzyme (e.g., heparan N-sulfatase (HNS)) for alysosomal storage disease (e.g., Sanfilippo A Syndrome) can be directlyintroduced into the cerebrospinal fluid (CSF) of a subject in need oftreatment at a high concentration (e.g., greater than about 3 mg/ml, 4mg/ml, 5 mg/ml, 10 mg/ml or more) such that the enzyme effectively andextensively diffuses across various surfaces and penetrates variousregions across the brain, including deep brain regions. Moresurprisingly, the present inventors have demonstrated that such highprotein concentration delivery can be done using simple saline orbuffer-based formulations and without inducing substantial adverseeffects, such as severe immune response, in the subject. Therefore, thepresent invention provides a highly efficient, clinically desirable andpatient-friendly approach for direct CNS delivery for the treatmentvarious diseases and disorders that have CNS components, in particular,lysosomal storage diseases. The present invention represents asignificant advancement in the field of CNS targeting and enzymereplacement therapy.

As described in detail below, the present inventors have successfullydeveloped stable formulations for effective intrathecal (IT)administration of an heparan N-sulfatase (HNS) protein. It iscontemplated, however, that various stable formulations described hereinare generally suitable for CNS delivery of therapeutic agents, includingvarious other lysosomal enzymes. Indeed, stable formulations accordingto the present invention can be used for CNS delivery via varioustechniques and routes including, but not limited to, intraparenchymal,intracerebral, intravetricular cerebral (ICV), intrathecal (e.g.,IT-Lumbar, IT-cisterna magna) administrations and any other techniquesand routes for injection directly or indirectly to the CNS and/or CSF.

It is also contemplated that various stable formulations describedherein are generally suitable for CNS delivery of other therapeuticagents, such as therapeutic proteins including various replacementenzymes for lysosomal storage diseases. In some embodiments, areplacement enzyme can be a synthetic, recombinant, gene-activated ornatural enzyme.

In one aspect, the present invention provides stable formulations forintrathecal administration comprising a heparan N-sulfatase (HNS)protein, salt, a buffering agent and a polysorbate surfactant. In someembodiments, the HNS protein is present at a concentration ranging fromapproximately 1-300 mg/ml (e.g., 1-250 mg/ml, 1-200 mg/ml, 1-150 mg/ml,1-100 mg/ml, 1-50 mg/ml). In some embodiments, the HNS protein ispresent at or up to a concentration selected from 2 mg/ml, 3 mg/ml, 4mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 35mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90mg/ml, 100 mg/ml, 150 mg/ml, 200 mg/ml, 250 mg/ml, or 300 mg/ml.

In various embodiments, the present invention includes a stableformulation of any of the embodiments described herein, wherein the HNSprotein comprises an amino acid sequence of SEQ ID NO:1. In someembodiments, the HNS protein comprises an amino acid sequence at least60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO:1.In some embodiments, the stable formulation of any of the embodimentsdescribed herein includes a salt. In some embodiments, the salt is NaCl.In some embodiments, the NaCl is present as a concentration ranging fromapproximately 0-300 mM (e.g., 0-250 mM, 0-200 mM, 0-150 mM, 0-100 mM,0-75 mM, 0-50 mM, or 0-30 mM). In some embodiments, the NaCl is presentat a concentration ranging from approximately 135-155 mM. In someembodiments, the NaCl is present at a concentration of approximately 145mM.

In various embodiments, the present invention includes a stableformulation of any of the embodiments described herein, wherein thepolysorbate surfactant is selected from the group consisting ofpolysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 andcombination thereof. In some embodiments, the polysorbate surfactant ispolysorbate 20. In some embodiments, the polysorbate 20 is present at aconcentration ranging approximately 0-0.02%. In some embodiments, thepolysorbate 20 is present at a concentration of approximately 0.005%. Insome embodiments, the polysorbate 20 is present at a concentration ofapproximately 0.02%.

In various embodiments, the present invention includes a stableformulation of any of the embodiments described herein, wherein theformulation further comprises a buffering agent. In some embodiments,the buffering agent is selected from the group consisting of phosphate,acetate, histidine, succinate, Tris, and combinations thereof. In someembodiments, the buffering agent is phosphate. In some embodiments, thephosphate is present at a concentration no greater than 50 mM (e.g., nogreater than 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, or5 mM). In some embodiments, the phosphate is present at a concentrationno greater than 20 mM. In certain embodiments, the phosphate is presentat a concentration of approximately 5 mM. In various aspects theinvention includes a stable formulation of any of the embodimentsdescribed herein, wherein the formulation has a pH of approximately 3-8(e.g., approximately 4-7.5, 5-8, 5-7.5, 5-6.5, 5-7.5, 5.5-8.0, 5.5-7.7,5.5-6.5, 6-7.5, 6-7.0, or 6.5-7.5). In some embodiments, the formulationhas a pH of approximately 6.5-7.5 (e.g., 6.5, 6.7, 6.9, 7.0, 7.2, 7.3,or 7.5). In some embodiments, the formulation has a pH of approximately7.0.

In some embodiments, the formulation further comprises a stabilizingagent. In certain embodiments, the stabilizing agent is selected fromthe group consisting of sucrose, glucose, mannitol, sorbitol, PEG 4000,histidine, arginine, lysine, phospholipids and combination thereof. Incertain embodiments, the stabilizing agent is sucrose. In someembodiments, the sucrose is present at a concentration ranging fromapproximately 0-10%. In some embodiments, the sucrose is present at aconcentration ranging from approximately 0.5-2.0%. In certainembodiments, the stabilizing agent is glucose. In some embodiments, theglucose is present at a concentration ranging from approximately0.5-1.0%.

In various embodiments, the present invention includes stableformulations of any of the embodiments described herein, wherein theformulation is a liquid formulation. In various embodiments, the presentinvention includes stable formulation of any of the embodimentsdescribed herein, wherein the formulation is formulated as lyophilizeddry powder.

In some embodiments, the present invention includes a stable formulationfor intrathecal administration comprising a heparan N-sulfatase (HNS)protein at a concentration up to approximately 30 mg/ml, NaCl at aconcentration of approximately 100-200 mM, polysorbate 20 at aconcentration of approximately 0.02%, phosphate at a concentration ofapproximately 5 mM, and a pH of approximately 7.0. In some embodiments,the HNS protein is at a concentration of approximately 15 mg/ml. In someembodiments, the HNS protein is at a concentration of approximately 30mg/ml, 40 mg/ml, 50 mg/ml, 100 mg/ml, 150 mg/ml, 200 mg/ml, 250 mg/ml,or 300 mg/ml. In some embodiments, the NaCl is at a concentration ofapproximately 145 mM. In some embodiments, the formulation furthercomprises sucrose at a concentration of approximately 0-10% (e.g.,approximately 0-5%, 1-7%, 1-2.5%. 1-1.5%, or 0.5-1.5%).

In some embodiments, the present invention includes a stable formulationfor intrathecal administration comprising a heparan N-sulfatase (HNS)protein at a concentration up to approximately 30 mg/ml, NaCl at aconcentration of approximately 145 mM, polysorbate 20 at a concentrationof approximately 0.02%, phosphate at a concentration of approximately 5mM, sucrose at a concentration of approximately 0.5-2%, and a pH ofapproximately 7.0.

In some embodiments, the present invention includes a stable formulationfor intrathecal administration comprising a heparan N-sulfatase (HNS)protein at a concentration up to approximately 30 mg/ml, NaCl at aconcentration of approximately 145 mM, polysorbate 20 at a concentrationof approximately 0.02%, phosphate at a concentration of approximately 5mM, glucose at a concentration of approximately 0.5-1.0%, and a pH ofapproximately 7.0.

In various aspects, the present invention includes a containercomprising a single dosage form of a stable formulation in variousembodiments described herein. In some embodiments, the container isselected from an ampule, a vial, a bottle, a cartridge, a reservoir, alyo-ject, or a pre-filled syringe. In some embodiments, the container isa pre-filled syringe. In some embodiments, the pre-filled syringe isselected from borosilicate glass syringes with baked silicone coating,borosilicate glass syringes with sprayed silicone, or plastic resinsyringes without silicone. In some embodiments, the stable formulationis present in a volume of less than about 50 mL (e.g., less than about45 ml, 40 ml, 35 ml, 30 ml, 25 ml, 20 ml, 15 ml, 10 ml, 5 ml, 4 ml, 3ml, 2.5 ml, 2.0 ml, 1.5 ml, 1.0 ml, or 0.5 ml). In some embodiments, thestable formulation is present in a volume of less than about 3.0 mL.

In various aspects, the present invention includes methods of treatingSanfilippo A Syndrome including the step of administering intrathecallyto a subject in need of treatment a formulation according to any of theembodiments described herein.

In some embodiments, the present invention includes a method of treatingSanfilippo A Syndrome including a step of administering intrathecally toa subject in need of treatment a formulation comprising an HNS proteinat a concentration ranging from approximately 1-300 mg/ml, NaCl at aconcentration of approximately 145 mM, polysorbate 20 at a concentrationof approximately 0.02%, and a pH of approximately 7.

In some embodiments, the intrathecal administration results in nosubstantial adverse effects (e.g., severe immune response) in thesubject. In some embodiments, the intrathecal administration results inno substantial adaptive T cell-mediated immune response in the subject.

In some embodiments, the intrathecal administration of the formulationresults in delivery of the HNS protein to various target tissues in thebrain, the spinal cord, and/or peripheral organs. In some embodiments,the intrathecal administration of the formulation results in delivery ofthe HNS protein to target brain tissues. In certain embodiments, the oneor more target brain tissues are selected from the group consisting oftissues from gray matter, white matter, periventricular areas,pia-arachnoid, meninges, neocortex, cerebellum, deep tissues in cerebralcortex, molecular layer, caudate/putamen region, midbrain, deep regionsof the pons or medulla, and combinations thereof. In certainembodiments, the HNS protein is delivered to neurons, glial cells,perivascular cells and/or meningeal cells. In some embodiments, the HNSprotein is further delivered to the neurons in the spinal cord.

In some embodiments, the intrathecal administration of the formulationfurther results in systemic delivery of the HNS protein in peripheraltarget tissues. In some embodiments, the peripheral target tissues areselected from liver, kidney, spleen and/or heart.

In some embodiments, the intrathecal administration of the formulationresults in lysosomal localization in brain target tissues, spinal cordneurons and/or peripheral target tissues. In some embodiments, theintrathecal administration of the formulation results in reduction ofGAG storage in the brain target tissues, spinal cord neurons and/orperipheral target tissues. In some embodiments, the GAG storage isreduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold,1.5-fold, or 2-fold as compared to a control (e.g., the pre-treatmentGAG storage in the subject). In some embodiments, the intrathecaladministration of the formulation results in reduced vacuolization inneurons (e.g., by at least 20%, 40%, 50%, 60%, 80%, 90%, 1-fold,1.5-fold, or 2-fold as compared to a control). In some embodiments, theneurons comprises Purkinje cells.

In some embodiments, the intrathecal administration of the formulationresults in increased HNS enzymatic activity in the brain target tissues,spinal cord neurons and/or peripheral target tissues. In someembodiments, the HNS enzymatic activity is increased by at least 1-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or10-fold as compared to a control (e.g., the pre-treatment endogenousenzymatic activity in the subject). In some embodiments, the increasedHNS enzymatic activity is at least approximately 10 nmol/hr/mg, 20nmol/hr/mg, 40 nmol/hr/mg, 50 nmol/hr/mg, 60 nmol/hr/mg, 70 nmol/hr/mg,80 nmol/hr/mg, 90 nmol/hr/mg, 100 nmol/hr/mg, 150 nmol/hr/mg, 200nmol/hr/mg, 250 nmol/hr/mg, 300 nmol/hr/mg, 350 nmol/hr/mg, 400nmol/hr/mg, 450 nmol/hr/mg, 500 nmol/hr/mg, 550 nmol/hr/mg or 600nmol/hr/mg.

In some embodiments, the HNS enzymatic activity is increased in thelumbar region. In some embodiments, the increased HNS enzymatic activityin the lumbar region is at least approximately 2000 nmol/hr/mg, 3000nmol/hr/mg, 4000 nmol/hr/mg, 5000 nmol/hr/mg, 6000 nmol/hr/mg, 7000nmol/hr/mg, 8000 nmol/hr/mg, 9000 nmol/hr/mg, or 10,000 nmol/hr/mg.

In some embodiments, the intrathecal administration of the formulationresults in reduced intensity, severity, or frequency, or delayed onsetof at least one symptom or feature of the Sanfilippo A Syndrome. In someembodiments, the at least one symptom or feature of the Sanfilippo ASyndrome is hearing loss, delayed speech development, deficits in motorskills, hyperactivity, mental retardation, aggressiveness and/or sleepdisturbances.

In some embodiments, the intrathecal administration takes place onceevery two weeks. In some embodiments, the intrathecal administrationtakes place once every month. In some embodiments, the intrathecaladministration takes place once every two months. In some embodiments,the intrathecal administration is used in conjunction with intravenousadministration. In some embodiments, the intravenous administration isno more frequent than once every week. In some embodiments, theintravenous administration is no more frequent than once every twoweeks. In some embodiments, the intravenous administration is no morefrequent than once every month. In some embodiments, the intravenousadministration is no more frequent than once every two months. Incertain embodiments, the intraveneous administration is more frequentthan monthly administration, such as twice weekly, weekly, every otherweek, or twice monthly.

In some embodiments, intraveneous and intrathecal administrations areperformed on the same day. In some embodiments, the intraveneous andintrathecal administrations are not performed within a certain amount oftime of each other, such as not within at least 2 days, within at least3 days, within at least 4 days, within at least 5 days, within at least6 days, within at least 7 days, or within at least one week. In someembodiments, intraveneous and intrathecal administrations are performedon an alternating schedule, such as alternating administrations weekly,every other week, twice monthly, or monthly. In some embodiments, anintrathecal administration replaces an intravenous administration in anadministration schedule, such as in a schedule of intraveneousadministration weekly, every other week, twice monthly, or monthly,every third or fourth or fifth administration in that schedule can bereplaced with an intrathecal administration in place of an intraveneousadministration.

In some embodiments, intraveneous and intrathecal administrations areperformed sequentially, such as performing intraveneous administrationsfirst (e.g., weekly, every other week, twice monthly, or monthly dosingfor two weeks, a month, two months, three months, four months, fivemonths, six months, a year or more) followed by IT administrations (e.g,weekly, every other week, twice monthly, or monthly dosing for more thantwo weeks, a month, two months, three months, four months, five months,six months, a year or more). In some embodiments, intrathecaladministrations are performed first (e.g., weekly, every other week,twice monthly, monthly, once every two months, once every three monthsdosing for two weeks, a month, two months, three months, four months,five months, six months, a year or more) followed by intraveneousadministrations (e.g., weekly, every other week, twice monthly, ormonthly dosing for more than two weeks, a month, two months, threemonths, four months, five months, six months, a year or more).

In some embodiments, the intrathecal administration is used in absenceof intravenous administration.

In some embodiments, the intrathecal administration is used in absenceof concurrent immunosuppressive therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are for illustration purposes only, not for limitation.

FIG. 1A-D depicts exemplary chromatograms of SEC-HPLC elution profilesfor HNS. FIG. 1A Profile of 2 mg/ml rhHNS 20 mM Citrate, pH 7.0; FIG. 1BScaled chromatogram of 2 mg/ml rhHNS 20 mM Citrate, pH 7.0, baseline(ilc); FIG. 1C Scaled chromatogram of 2 mg/ml rhHNS 20 mM Citrate, pH7.0 after 7 days at 50° C.; FIG. 1D Overlay of the wavelength scan ofthe 16 min peak and 26 min dimer peak of 2 mg/ml Citrate, pH 7.0 after 7days at 50° C.

FIG. 2A-C depicts exemplary reduced SDS-PAGE gels from pH studies forrhHNS in various buffers and at various pH.

FIG. 3A-C depicts exemplary non-reduced SDS-PAGE gels from pH studiesfor rhHNS in various buffers and at various pH.

FIG. 4 depicts exemplary pH-dependent thermal stability in citrate asdetermined by DSC. The highest melting temperature of rhHNS in citratewas 90° C. at pH 6.0. The melting temperature of rhHNS at every pHexamined exceeds 70° C.

FIG. 5 depicts exemplary pH-dependent thermal stability in phosphate asdetermined by DSC. rhHNS formulations containing phosphate showedgreatest thermal stability at pH 6-7. The melting temperature of rhHNSat every pH examined exceeds 70° C.

FIG. 6A-B depicts exemplary silver stained SDS-PAGE gels of rhHNSformulations from ionic effects study after 7 days at 50° C. Gels wererun using samples which were boiled for 10 minutes.

FIG. 7A-B depicts an exemplary rhHNS solubility study. FIG. 7A Effect ofpH on rhHNS solubility; FIG. 7B Effect of salt concentration on rhHNSsolubility. Increasing pH and sodium chloride appear to increase rhHNSsolubility.

FIG. 8A-B depicts an exemplary study of the effect of salt on nativestate of rhHNS using AUC. FIG. 8A Effect of 145 mM salt; FIG. 8B Effectof 300 mM salt.

FIG. 9A and FIG. 9B depicts an exemplary study on the effect of sucroselevel and lyophilization unit on cake appearance of lyophilized rhHNSformulations. FIG. 9A VirTis lyo unit; upper panel, 1% sucrose; lowerpanel 1.5% sucrose; FIG. 9B 1.5% sucrose; upper panel, VirTis lyo unit;lower panel LyoStar lyo unit.

FIG. 10 depicts exemplary particulate images by Micro-Flow Imaging (MFI)for lyophilized rhHNS samples.

FIG. 11 depicts exemplary images of a study of the effect of polysorbate20 on particulates detected by MFI for pre-lyophilized rhHNS samplescontaining 1.5% sucrose after 0.22 μm filtration.

FIG. 12A depicts an exemplary result illustrating CSF concentrations ofrhHNS as a function of time at 1.5, 4.5 and 8.3 mg doses following 6months of dosing. FIG. 12B details an exemplary result illustratingAnti-HNS antibody concentrations in the CSF after 6 months of ITadministration of 1.5, 4.5 and 8.3 mg doses in monkeys. Data are shownfor male and females combined. FIG. 12C details an exemplary resultillustrating Anti-HNS antibody concentrations in the CSF after 6 monthsof IT administration of 1.5, 4.5 and 8.3 mg doses in monkeys following 6months of dosing. Data are shown for male and females combined. The twohighest concentrations (32,205 ng/mL and 15,467 ng/mL) post IT dose 6 at8.3 mg of rhHNS were excluded from the plot because no CSF samples weretaken predose 6.

FIG. 13A-F depicts exemplary representative images of tissue sectionsfrom the meninges and parenchyma of the brain stained with hematoxylinand eosin. FIG. 13A depicts an exemplary result illustrating a low-powerview of neutrophilic infiltrates local to the IT catheter in a DCmonkey. FIG. 13B depicts an exemplary result illustrating a high-powerview of eosinophilic infiltrates in the meninges of a high-dose (8.3mg/dose) monkey; the overall severity of infiltrates was similar to themid-dose (4.5 mg/dose) group (not shown). FIG. 13C depicts an exemplaryresult illustrating a high-power view of a low-dose (1.5 mg/dose) monkeyshowing eosinophils in the perivascular space (brain parenchyma). FIG.13D depicts an exemplary result illustrating a low-dose monkey (1.5mg/dose) showing eosinophils in the perivascular space and adjoiningparenchyma. FIG. 13E depicts an exemplary result illustratingeosinophils in the spinal cord parenchyma (indicated by arrows) of alow-dose group animal; neurons in the area are normal. FIG. 13F depictsan exemplary result illustrating eosinophils and an area of microgliosis(arrows indicate eosinophils; the box indicates an area of microgliosis)in a low-dose (1.5 mg/dose) monkey. There are several large neurons inthe area, all of which are normal. Scale bars: 200 μm.

FIG. 14A-D depicts an exemplary result illustrating rhHNS enzymeactivity in monkey spinal cords and brains. FIGS. 14A and 14B depict anexemplary result illustrating activity in the spinal cords of (A) maleand (B) female monkeys. Slice—3=lumbar, slices 3, 6=thoracic, and slice9=cervical; 0=catheter tip. FIGS. 14C and 14D depicts an exemplaryresult illustrating rhHNS activity in the brains of (C) male and (D)female monkeys. Slices are numbered rostral to caudal (3 to 15). Alltissue samples were collected approximately 24 hours after the last doseor 4 weeks after the last dose for the recovery animals. DC, devicecontrol. The data represent mean±SEM for n=4 monkeys per treatmentgroup.

FIGS. 15A and 15B depicts an exemplary result illustrating enzymeactivity in monkey brain and liver. FIG. 15A depicts an exemplary resultillustrating rhHNS activity distribution in the high-dose (8.3 mg/dose)group monkey brain. The fold-change in activity for surface, deep, andvery deep (periventricular) areas of the brain compared with endogenouslevels (DC group) is shown. All tissue samples were collectedapproximately 24 hours after the last dose or 4 weeks after the lastdose for the recovery animals. The data represent mean±SEM for n=6monkeys (both sexes), brain slices 6 and 9. Data for two monkeys werenot included; at necropsy the catheters were not found to be patent.FIG. 15B shows rhHNS activity in monkey liver. All tissue samples werecollected approximately 24 hours after the last dose or 4 weeks afterthe last dose for the recovery animals. DC, device control. Rec,recovery. The data represent mean±SEM for n=4 monkeys per treatmentgroup except for the low-dose (4.5 mg/dose) female group (n=3).

FIG. 16A-D depicts an exemplary result illustrating rhHNS localizationin juvenile cynomolgus monkey cerebellum: 3-month interim cohort. FIG.16A depicts an exemplary result illustrating cerebellum of a vehiclecontrol animal (0 mg/dose) negative for rhHNS immunostaining; 20×magnification. FIG. 16B depicts an exemplary result illustratingcerebellum of a low-dose (1.5 mg/dose) animal showing minimal positivestaining limited to the molecular layer; 20× magnification. FIG. 16Cdepicts an exemplary result illustrating cerebellum of a mid-dose (4.5mg/dose) animal showing minimal staining in the outer granular layer;20× magnification. FIG. 16D depicts an exemplary result illustratingmoderate staining in the cerebellum of a high-dose (8.3 mg/dose) animalincluding molecular, outer granular layer, and Purkinje cells; 20×magnification.

FIG. 17 depicts an exemplary study of the concentration of rhHNS in thehead region plotted with time in the first 20 minutes after IT dosing of¹²⁴I-HNS at 1 and 10 mg/kg.

FIG. 18 depicts an exemplary study of the concentration of rhHNS in thebrain plotted with time after IT dosing of ¹²⁴I-HNS at 1 and 10 mg/kg.

FIG. 19 depicts an exemplary study of the concentration of rhHNS in thebrain region plotted with time after IT dosing of ¹²⁴I-HNS at 1 and 10mg/kg.

FIG. 20 depicts an exemplary study of the concentration of rhHNS in thehead region plotted with time after IT dosing of ¹²⁴I-HNS at 1 and 10mg/kg.

FIG. 21 depicts an exemplary study of the concentration of rhHNS in theproximal spine plotted with time after IT dosing of ¹²⁴I-HNS at 1 and 10mg/kg.

FIG. 22 depicts an exemplary study of the concentration of rhHNS in themid-spine plotted with time after IT dosing of ¹²⁴I-HNS at 1 and 10mg/kg.

FIG. 23 depicts an exemplary study of the concentration of rhHNS in thedistal spine plotted with time after IT dosing of ¹²⁴I-HNS at 1 and 10mg/kg.

FIG. 24 depicts an exemplary study of the concentration of rhHNS in theliver plotted with time after IT dosing of ¹²⁴I-HNS at 1 and 10 mg/kg.

FIG. 25 depicts an exemplary study of the concentration of rhHNS in thebrain plotted with time after IT dosing of ¹²⁴I-HNS at 1 and 10 mg/kg,individual (top) and mean±SD (bottom).

FIG. 26 depicts an exemplary study of the hepatic concentration of rhHNSplotted with time after IT dosing of ¹²⁴I-HNS at 1 and 10 mg/kg,individual (top) and mean±SD (bottom).

FIG. 27 depicts an exemplary study of the renal concentration of rhHNSplotted with time after IT dosing of ¹²⁴I-HNS at 1 and 10 mg/kg,individual (top) and mean±SD (bottom).

FIG. 28 depicts an exemplary study of the heart concentration of rhHNSplotted with time after IT dosing of ¹²⁴I-HNS at 1 and 10 mg/kg,individual (top) and mean±SD (bottom).

FIG. 29 depicts an exemplary study of the skin concentration of rhHNSplotted with time after IT dosing of ¹²⁴I-HNS at 1 and 10 mg/kg,individual (top) and mean±SD (bottom).

FIG. 30 depicts an exemplary study of the brain concentration of rhHNSplotted with time after IT dosing of ¹²⁴I-HNS at 1 and 10 mg/kg (top),and a comparison of the non-compartmental PK parameters in the brain(bottom).

FIG. 31 depicts an exemplary study of the liver concentration of rhHNSplotted with time after IT dosing of ¹²⁴I-HNS at 1 and 10 mg/kg (top),and a comparison of the non-compartmental PK parameters in the liver(bottom).

FIG. 32 depicts an exemplary intrathecal drug delivery device (IDDD).

FIG. 33 depicts an exemplary port-a-cath low profile intrathecalimplantable access system.

FIG. 34 depicts an exemplary intrathecal drug delivery device (IDDD).

FIG. 35 depicts an exemplary intrathecal drug delivery device (IDDD),which allows for in-home administration for CNS enzyme replacementtherapy (ERT).

FIG. 36 illustrates an exemplary diagram of an intrathecal drug deliverydevice (IDDD) with a securing mechanism.

FIG. 37A depicts exemplary locations within a patient's body where anIDDD may be placed; FIG. 37B depicts various components of anintrathecal drug delivery device (IDDD); and FIG. 37C depicts anexemplary insertion location within a patient's body for IT-lumbarinjection.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Amelioration: As used herein, the term “amelioration” is meant theprevention, reduction or palliation of a state, or improvement of thestate of a subject. Amelioration includes, but does not require completerecovery or complete prevention of a disease condition. In someembodiments, amelioration includes increasing levels of relevant proteinor its activity that is deficient in relevant disease tissues.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any agent that has activity in abiological system, and particularly in an organism. For instance, anagent that, when administered to an organism, has a biological effect onthat organism, is considered to be biologically active. In particularembodiments, where a protein or polypeptide is biologically active, aportion of that protein or polypeptide that shares at least onebiological activity of the protein or polypeptide is typically referredto as a “biologically active” portion.

Bulking agent: As used herein, the term “bulking agent” refers to acompound which adds mass to the lyophilized mixture and contributes tothe physical structure of the lyophilized cake (e.g., facilitates theproduction of an essentially uniform lyophilized cake which maintains anopen pore structure). Exemplary bulking agents include mannitol,glycine, sodium chloride, hydroxyethyl starch, lactose, sucrose,trehalose, polyethylene glycol and dextran.

Cation-independent mannose-6-phosphate receptor (CI-MPR): As usedherein, the term “cation-independent mannose-6-phosphate receptor(CI-MPR)” refers to a cellular receptor that binds mannose-6-phosphate(M6P) tags on acid hydrolase precursors in the Golgi apparatus that aredestined for transport to the lysosome. In addition tomannose-6-phosphates, the CI-MPR also binds other proteins includingIGF-II. The CI-MPR is also known as “M6P/IGF-II receptor,”“CI-MPR/IGF-II receptor,” “IGF-II receptor” or “IGF2 Receptor.” Theseterms and abbreviations thereof are used interchangeably herein.

Concurrent immunosuppressant therapy: As used herein, the term“concurrent immunosuppressant therapy” includes any immunosuppressanttherapy used as pre-treatment, preconditioning or in parallel to atreatment method.

Diluent: As used herein, the term “diluent” refers to a pharmaceuticallyacceptable (e.g., safe and non-toxic for administration to a human)diluting substance useful for the preparation of a reconstitutedformulation. Exemplary diluents include sterile water, bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution.

Dosage form: As used herein, the terms “dosage form” and “unit dosageform” refer to a physically discrete unit of a therapeutic protein forthe patient to be treated. Each unit contains a predetermined quantityof active material calculated to produce the desired therapeutic effect.It will be understood, however, that the total dosage of the compositionwill be decided by the attending physician within the scope of soundmedical judgment.

Enzyme replacement therapy (ERT): As used herein, the term “enzymereplacement therapy (ERT)” refers to any therapeutic strategy thatcorrects an enzyme deficiency by providing the missing enzyme. In someembodiments, the missing enzyme is provided by intrathecaladministration. In some embodiments, the missing enzyme is provided byinfusing into bloodstream. Once administered, enzyme is taken up bycells and transported to the lysosome, where the enzyme acts toeliminate material that has accumulated in the lysosomes due to theenzyme deficiency. Typically, for lysosomal enzyme replacement therapyto be effective, the therapeutic enzyme is delivered to lysosomes in theappropriate cells in target tissues where the storage defect ismanifest.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control individual (or multiple controlindividuals) in the absence of the treatment described herein. A“control individual” is an individual afflicted with the same form oflysosomal storage disease as the individual being treated, who is aboutthe same age as the individual being treated (to ensure that the stagesof the disease in the treated individual and the control individual(s)are comparable).

Individual, subject, patient: As used herein, the terms “subject,”“individual” or “patient” refer to a human or a non-human mammaliansubject. The individual (also referred to as “patient” or “subject”)being treated is an individual (fetus, infant, child, adolescent, oradult human) suffering from a disease.

Intrathecal administration: As used herein, the term “intrathecaladministration” or “intrathecal injection” refers to an injection intothe spinal canal (intrathecal space surrounding the spinal cord).Various techniques may be used including, without limitation, lateralcerebroventricular injection through a burrhole or cisternal or lumbarpuncture or the like. In some embodiments, “intrathecal administration”or “intrathecal delivery” according to the present invention refers toIT administration or delivery via the lumbar area or region, i.e.,lumbar IT administration or delivery. As used herein, the term “lumbarregion” or “lumbar area” refers to the area between the third and fourthlumbar (lower back) vertebrae and, more inclusively, the L2-S1 region ofthe spine.

Linker: As used herein, the term “linker” refers to, in a fusionprotein, an amino acid sequence other than that appearing at aparticular position in the natural protein and is generally designed tobe flexible or to interpose a structure, such as an a-helix, between twoprotein moieties. A linker is also referred to as a spacer.

Lyoprotectant: As used herein, the term “lyoprotectant” refers to amolecule that prevents or reduces chemical and/or physical instabilityof a protein or other substance upon lyophilization and subsequentstorage. Exemplary lyoprotectants include sugars such as sucrose ortrehalose; an amino acid such as monosodium glutamate or histidine; amethylamine such as betaine; a lyotropic salt such as magnesium sulfate:a polyol such as trihydric or higher sugar alcohols, e.g. glycerin,erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol;propylene glycol; polyethylene glycol; Pluronics; and combinationsthereof. In some embodiments, a lyoprotectant is a non-reducing sugar,such as trehalose or sucrose.

Lysosomal enzyme: As used herein, the term “lysosomal enzyme” refers toany enzyme that is capable of reducing accumulated materials inmammalian lysosomes or that can rescue or ameliorate one or morelysosomal storage disease symptoms. Lysosomal enzymes suitable for theinvention include both wild-type or modified lysosomal enzymes and canbe produced using recombinant and synthetic methods or purified fromnature sources. Exemplary lysosomal enzymes are listed in Table 1.

Lysosomal enzyme deficiency: As used herein, “lysosomal enzymedeficiency” refers to a group of genetic disorders that result fromdeficiency in at least one of the enzymes that are required to breakmacromolecules (e.g., enzyme substartes) down to peptides, amino acids,monosaccharides, nucleic acids and fatty acids in lysosomes. As aresult, individuals suffering from lysosomal enzyme deficiencies haveaccumulated materials in various tissues (e.g., CNS, liver, spleen, gut,blood vessel walls and other organs).

Lysosomal Storage Disease: As used herein, the term “lysosomal storagedisease” refers to any disease resulting from the deficiency of one ormore lysosomal enzymes necessary for metabolizing naturalmacromolecules. These diseases typically result in the accumulation ofun-degraded molecules in the lysosomes, resulting in increased numbersof storage granules (also termed storage vesicles). These diseases andvarious examples are described in more detail below.

Polypeptide: As used herein, a “polypeptide”, generally speaking, is astring of at least two amino acids attached to one another by a peptidebond. In some embodiments, a polypeptide may include at least 3-5 aminoacids, each of which is attached to others by way of at least onepeptide bond. Those of ordinary skill in the art will appreciate thatpolypeptides sometimes include “non-natural” amino acids or otherentities that nonetheless are capable of integrating into a polypeptidechain, optionally.

Replacement enzyme: As used herein, the term “replacement enzyme” refersto any enzyme that can act to replace at least in part the deficient ormissing enzyme in a disease to be treated. In some embodiments, the term“replacement enzyme” refers to any enzyme that can act to replace atleast in part the deficient or missing lysosomal enzyme in a lysosomalstorage disease to be treated. In some embodiments, a replacement enzymeis capable of reducing accumulated materials in mammalian lysosomes orthat can rescue or ameliorate one or more lysosomal storage diseasesymptoms. Replacement enzymes suitable for the invention include bothwild-type or modified lysosomal enzymes and can be produced usingrecombinant and synthetic methods or purified from nature sources. Areplacement enzyme can be a recombinant, synthetic, gene-activated ornatural enzyme.

Soluble: As used herein, the term “soluble” refers to the ability of atherapeutic agent to form a homogenous solution. In some embodiments,the solubility of the therapeutic agent in the solution into which it isadministered and by which it is transported to the target site of action(e.g., the cells and tissues of the brain) is sufficient to permit thedelivery of a therapeutically effective amount of the therapeutic agentto the targeted site of action. Several factors can impact thesolubility of the therapeutic agents. For example, relevant factorswhich may impact protein solubility include ionic strength, amino acidsequence and the presence of other co-solubilizing agents or salts(e.g., calcium salts). In some embodiments, the pharmaceuticalcompositions are formulated such that calcium salts are excluded fromsuch compositions. In some embodiments, therapeutic agents in accordancewith the present invention are soluble in its correspondingpharmaceutical composition. It will be appreciated that, while isotonicsolutions are generally preferred for parenterally administered drugs,the use of isotonic solutions may limit adequate solubility for sometherapeutic agents and, in particular some proteins and/or enzymes.Slightly hypertonic solutions (e.g., up to 175 mM sodium chloride in 5mM sodium phosphate at pH 7.0) and sugar-containing solutions (e.g., upto 2% sucrose in 5 mM sodium phosphate at pH 7.0) have been demonstratedto be well tolerated in monkeys. For example, the most common approvedCNS bolus formulation composition is saline (150 mM NaCl in water).

Stability: As used herein, the term “stable” refers to the ability ofthe therapeutic agent (e.g., a recombinant enzyme) to maintain itstherapeutic efficacy (e.g., all or the majority of its intendedbiological activity and/or physiochemical integrity) over extendedperiods of time. The stability of a therapeutic agent, and thecapability of the pharmaceutical composition to maintain stability ofsuch therapeutic agent, may be assessed over extended periods of time(e.g., for at least 1, 3, 6, 12, 18, 24, 30, 36 months or more). Ingeneral, pharmaceutical compositions described herein have beenformulated such that they are capable of stabilizing, or alternativelyslowing or preventing the degradation, of one or more therapeutic agentsformulated therewith (e.g., recombinant proteins). In the context of aformulation a stable formulation is one in which the therapeutic agenttherein essentially retains its physical and/or chemical integrity andbiological activity upon storage and during processes (such asfreeze/thaw, mechanical mixing and lyophilization). For proteinstability, it can be measure by formation of high molecular weight (HMW)aggregates, loss of enzyme activity, generation of peptide fragments andshift of charge profiles.

Subject: As used herein, the term “subject” means any mammal, includinghumans. In certain embodiments of the present invention the subject isan adult, an adolescent or an infant. Also contemplated by the presentinvention are the administration of the pharmaceutical compositionsand/or performance of the methods of treatment in-utero.

Substantial homology: The phrase “substantial homology” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially homologous” ifthey contain homologous residues in corresponding positions. Homologousresidues may be identical residues. Alternatively, homologous residuesmay be non-identical residues will appropriately similar structuraland/or functional characteristics. For example, as is well known bythose of ordinary skill in the art, certain amino acids are typicallyclassified as “hydrophobic” or “hydrophilic” amino acids, and/or ashaving “polar” or “non-polar” side chains Substitution of one amino acidfor another of the same type may often be considered a “homologous”substitution.

As is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul, et al.,Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990;Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLASTand PSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al.,Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins,Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods andProtocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999.In addition to identifying homologous sequences, the programs mentionedabove typically provide an indication of the degree of homology. In someembodiments, two sequences are considered to be substantially homologousif at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues arehomologous over a relevant stretch of residues. In some embodiments, therelevant stretch is a complete sequence. In some embodiments, therelevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500 or more residues.

Substantial identity: The phrase “substantial identity” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially identical” ifthey contain identical residues in corresponding positions. As is wellknown in this art, amino acid or nucleic acid sequences may be comparedusing any of a variety of algorithms, including those available incommercial computer programs such as BLASTN for nucleotide sequences andBLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplarysuch programs are described in Altschul, et al., Basic local alignmentsearch tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al.,Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402,1997; Baxevanis et al., Bioinformatics: A Practical Guide to theAnalysis of Genes and Proteins, Wiley, 1998; and Misener, et al.,(eds.), Bioinformatics Methods and Protocols (Methods in MolecularBiology, Vol. 132), Humana Press, 1999. In addition to identifyingidentical sequences, the programs mentioned above typically provide anindication of the degree of identity. In some embodiments, two sequencesare considered to be substantially identical if at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more of their corresponding residues are identical over arelevant stretch of residues. In some embodiments, the relevant stretchis a complete sequence. In some embodiments, the relevant stretch is atleast 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500 or more residues.

Synthetic CSF: As used herein, the term “synthetic CSF” refers to asolution that has pH, electrolyte composition, glucose content andosmolarity consistent with the cerebrospinal fluid. Synthetic CSF isalso referred to as artificial CSF. In some embodiments, synthetic CSFis an Elliott's B solution.

Suitable for CNS delivery: As used herein, the phrase “suitable for CNSdelivery” or “suitable for intrathecal delivery” as it relates to thepharmaceutical compositions of the present invention generally refers tothe stability, tolerability, and solubility properties of suchcompositions, as well as the ability of such compositions to deliver aneffective amount of the therapeutic agent contained therein to thetargeted site of delivery (e.g., the CSF or the brain).

Target tissues: As used herein, the term “target tissues” refers to anytissue that is affected by the lysosomal storage disease to be treatedor any tissue in which the deficient lysosomal enzyme is normallyexpressed. In some embodiments, target tissues include those tissues inwhich there is a detectable or abnormally high amount of enzymesubstrate, for example stored in the cellular lysosomes of the tissue,in patients suffering from or susceptible to the lysosomal storagedisease. In some embodiments, target tissues include those tissues thatdisplay disease-associated pathology, symptom, or feature. In someembodiments, target tissues include those tissues in which the deficientlysosomal enzyme is normally expressed at an elevated level. As usedherein, a target tissue may be a brain target tissue, a spinal cordtarget tissue an/or a peripheral target tissue. Exemplary target tissuesare described in detail below.

Therapeutic moiety: As used herein, the term “therapeutic moiety” refersto a portion of a molecule that renders the therapeutic effect of themolecule. In some embodiments, a therapeutic moiety is a polypeptidehaving therapeutic activity.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” refers to an amount of a therapeuticprotein (e.g., replacement enzyme) which confers a therapeutic effect onthe treated subject, at a reasonable benefit/risk ratio applicable toany medical treatment. The therapeutic effect may be objective (i.e.,measurable by some test or marker) or subjective (i.e., subject gives anindication of or feels an effect). In particular, the “therapeuticallyeffective amount” refers to an amount of a therapeutic protein orcomposition effective to treat, ameliorate, or prevent a desired diseaseor condition, or to exhibit a detectable therapeutic or preventativeeffect, such as by ameliorating symptoms associated with the disease,preventing or delaying the onset or progression of the disease, and/oralso lessening the severity or frequency of symptoms of the disease. Atherapeutically effective amount is commonly administered in a dosingregimen that may comprise multiple unit doses. For any particulartherapeutic protein, a therapeutically effective amount (and/or anappropriate unit dose within an effective dosing regimen) may vary, forexample, depending on route of administration, on combination with otherpharmaceutical agents. Also, the specific therapeutically effectiveamount (and/or unit dose) for any particular patient may depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific pharmaceutical agentemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and/or rate of excretion or metabolism of thespecific fusion protein employed; the duration of the treatment; andlike factors as is well known in the medical arts.

Tolerable: As used herein, the terms “tolerable” and “tolerability”refer to the ability of the pharmaceutical compositions of the presentinvention to not elicit an adverse reaction in the subject to whom suchcomposition is administered, or alternatively not to elicit a seriousadverse reaction in the subject to whom such composition isadministered. In some embodiments, the pharmaceutical compositions ofthe present invention are well tolerated by the subject to whom suchcompositions is administered.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a therapeutic protein (e.g.,lysosomal enzyme) that partially or completely alleviates, ameliorates,relieves, inhibits, delays onset of, reduces severity of and/or reducesincidence of one or more symptoms or features of a particular disease,disorder, and/or condition (e.g., Hunters syndrome, Sanfilippo Asyndrome, Sanfilippo B syndrome). Such treatment may be of a subject whodoes not exhibit signs of the relevant disease, disorder and/orcondition and/or of a subject who exhibits only early signs of thedisease, disorder, and/or condition. Alternatively or additionally, suchtreatment may be of a subject who exhibits one or more established signsof the relevant disease, disorder and/or condition.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, among other things, improved methods andcompositions for effective direct delivery of a therapeutic agent to thecentral nervous system (CNS). As discussed above, the present inventionis based on unexpected discovery that a replacement enzyme (e.g., an HNSprotein) for a lysosomal storage disease (e.g., Sanfilippo A Syndrome)can be directly introduced into the cerebrospinal fluid (CSF) of asubject in need of treatment at a high concentration without inducingsubstantial adverse effects in the subject. More surprisingly, thepresent inventors found that the replacement enzyme may be delivered ina simple saline or buffer-based formulation, without using syntheticCSF. Even more unexpectedly, intrathecal delivery according to thepresent invention does not result in substantial adverse effects, suchas severe immune response, in the subject. Therefore, in someembodiments, intrathecal delivery according to the present invention maybe used in absence of concurrent immunosuppressant therapy (e.g.,without induction of immune tolerance by pre-treatment orpre-conditioning).

In some embodiments, intrathecal delivery according to the presentinvention permits efficient diffusion across various brain tissuesresulting in effective delivery of the replacement enzyme in varioustarget brain tissues in surface, shallow and/or deep brain regions. Insome embodiments, intrathecal delivery according to the presentinvention resulted in sufficient amount of replacement enzymes enteringthe peripheral circulation. As a result, in some cases, intrathecaldelivery according to the present invention resulted in delivery of thereplacement enzyme in peripheral tissues, such as liver, heart, spleenand kidney. This discovery is unexpected and can be particular usefulfor the treatment of lysosomal storage diseases that have both CNS andperipheral components, which would typically require both regularintrathecal administration and intravenous administration. It iscontemplated that intrathecal delivery according to the presentinvention may allow reduced dosing and/or frequency of iv injectionwithout compromising therapeutic effects in treating peripheralsymptoms.

The present invention provides various unexpected and beneficialfeatures that allow efficient and convenient delivery of replacementenzymes to various brain target tissues, resulting in effectivetreatment of lysosomal storage diseases that have CNS indications.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

Replacement Enzymes

Heparan-N-Sulfatase (HNS) Protein

In some embodiments, inventive methods and compositions provided by thepresent invention are used to deliver an Heparan-N-Sulfatase (HNS)protein to the CNS for treatment of Sanfilippo A. A suitable HNS proteincan be any molecule or a portion of a molecule that can substitute fornaturally-occurring Heparan-N-Sulfatase (HNS) protein activity or rescueone or more phenotypes or symptoms associated with HNS-deficiency. Insome embodiments, a replacement enzyme suitable for the invention is apolypeptide having an N-terminus and a C-terminus and an amino acidsequence substantially similar or identical to mature human HNS protein.

Typically, human HNS is produced as a precursor molecule that isprocessed to a mature form. This process generally occurs by removingthe 20 amino acid signal peptide. Typically, the precursor form is alsoreferred to as full-length precursor or full-length HNS protein, whichcontains 502 amino acids. The N-terminal 20 amino acids are cleaved,resulting in a mature form that is 482 amino acids in length. Thus, itis contemplated that the N-terminal 20 amino acids is generally notrequired for the HNS protein activity. The amino acid sequences of themature form (SEQ ID NO:1) and full-length precursor (SEQ ID NO:2) of atypical wild-type or naturally-occurring human HNS protein are shown inTable 1.

TABLE 1 Human Heparan-N-Sulfatase Mature FormRPRNALLLLA DDGGFESGAY NNSAIATPHL DALARRSLLFRNAFTSVSSC SPSRASLLTG LPQHQNGMYG LHQDVHHFNSFDKVRSLPLL LSQAGVRTGI IGKKHVGPET VYPFDFAYTEENGSVLQVGR NITRIKLLVR KFLQTQDDRP FFLYVAFHDPHRCGHSQPQY GTFCEKFGNG ESGMGRIPDW TPQAYDPLDVLVPYFVPNTP AARADLAAQY TTVGRMDQGV GLVLQELRDAGVLNDTLVIF TSDNGIPFPS GRTNLYWPGT AEPLLVSSPEHPKRWGQVSE AYVSLLDLTP TILDWFSIPY PSYAIFGSKTIHLTGRSLLP ALEAEPLWAT VFGSQSHHEV TMSYPMRSVQHRHFRLVHNL NFKMPFPIDQ DFYVSPTFQD LLNRTTAGQPTGWYKDLRHY YYRARWELYD RSRDPHETQN LATDPRFAQLLEMLRDQLAK WQWETHDPWV CAPDGVLEEK LSPQCQPLHN EL (SEQ ID NO: 1)Full-Length MSCPVPACCA LLLVLGLCRA RPRNALLLLA DDGGFESGAY PrecursorNNSAIATPHL DALARRSLLF RNAFTSVSSC SPSRASLLTGLPQHQNGMYG LHQDVHHFNS FDKVRSLPLL LSQAGVRTGIIGKKHVGPET VYPFDFAYTE ENGSVLQVGR NITRIKLLVRKFLQTQDDRP FFLYVAFHDP HRCGHSQPQY GTFCEKFGNGESGMGRIPDW TPQAYDPLDV LVPYFVPNTP AARADLAAQYTTVGRMDQGV GLVLQELRDA GVLNDTLVIF TSDNGIPFPSGRTNLYWPGT AEPLLVSSPE HPKRWGQVSE AYVSLLDLTPTILDWFSIPY PSYAIFGSKT IHLTGRSLLP ALEAEPLWATVFGSQSHHEV TMSYPMRSVQ HRHFRLVHNL NFKMPFPIDQDFYVSPTFQD LLNRTTAGQP TGWYKDLRHY YYRARWELYDRSRDPHETQN LATDPRFAQL LEMLRDQLAK WQWETHDPWV CAPDGVLEEK LSPQCQPLHNEL (SEQ ID NO: 2)

Thus, in some embodiments, a therapeutic moiety suitable for the presentinvention is mature human HNS protein (SEQ ID NO:1). In someembodiments, a suitable therapeutic moiety may be a homologue or ananalogue of mature human HNS protein. For example, a homologue or ananalogue of mature human HNS protein may be a modified mature human HNSprotein containing one or more amino acid substitutions, deletions,and/or insertions as compared to a wild-type or naturally-occurring HNSprotein (e.g., SEQ ID NO:1), while retaining substantial HNS proteinactivity. Thus, in some embodiments, a therapeutic moiety suitable forthe present invention is substantially homologous to mature human HNSprotein (SEQ ID NO:1). In some embodiments, a therapeutic moietysuitable for the present invention has an amino acid sequence at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more homologous to SEQ ID NO:1. In someembodiments, a therapeutic moiety suitable for the present invention issubstantially identical to mature human HNS protein (SEQ ID NO:1). Insome embodiments, a therapeutic moiety suitable for the presentinvention has an amino acid sequence at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to SEQ ID NO:1. In some embodiments, a therapeutic moietysuitable for the present invention contains a fragment or a portion ofmature human HNS protein.

Alternatively, a therapeutic moiety suitable for the present inventionis full-length HNS protein. In some embodiments, a suitable therapeuticmoiety may be a homologue or an analogue of full-length human HNSprotein. For example, a homologue or an analogue of full-length humanHNS protein may be a modified full-length human HNS protein containingone or more amino acid substitutions, deletions, and/or insertions ascompared to a wild-type or naturally-occurring full-length HNS protein(e.g., SEQ ID NO:2), while retaining substantial HNS protein activity.Thus, In some embodiments, a therapeutic moiety suitable for the presentinvention is substantially homologous to full-length human HNS protein(SEQ ID NO:2). In some embodiments, a therapeutic moiety suitable forthe present invention has an amino acid sequence at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more homologous to SEQ ID NO:2. In some embodiments, atherapeutic moiety suitable for the present invention is substantiallyidentical to SEQ ID NO:2. In some embodiments, a therapeutic moietysuitable for the present invention has an amino acid sequence at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to SEQ ID NO:2. In someembodiments, a therapeutic moiety suitable for the present inventioncontains a fragment or a portion of full-length human HNS protein. Asused herein, a full-length HNS protein typically contains signal peptidesequence.

Other Lysosomal Storage Diseases and Replacement Enzymes

It is contemplated that inventive methods and compositions according tothe present invention can be used to treat other lysosomal storagediseases, in particular those lysosomal storage diseases having CNSetiology and/or symptoms, including, but are not limited to,aspartylglucosaminuria, cholesterol ester storage disease, Wolmandisease, cystinosis, Danon disease, Fabry disease, Farberlipogranulomatosis, Farber disease, fucosidosis, galactosialidosis typesI/II, Gaucher disease types I/II/III, globoid cell leukodystrophy,Krabbe disease, glycogen storage disease II, Pompe disease,GM1-gangliosidosis types I/II/III, GM2-gangliosidosis type I, Tay Sachsdisease, GM2-gangliosidosis type II, Sandhoff disease,GM2-gangliosidosis, α-mannosidosis types I/II, .beta.-mannosidosis,metachromatic leukodystrophy, mucolipidosis type I, sialidosis typesI/II, mucolipidosis types II/III, I-cell disease, mucolipidosis typeIIIC pseudo-Hurler polydystrophy, mucopolysaccharidosis type I,mucopolysaccharidosis type II, mucopolysaccharidosis type IIIA,Sanfilippo syndrome (e.g., types A, B, C, D), mucopolysaccharidosis typeIIIB, mucopolysaccharidosis type IIIC, mucopolysaccharidosis type IIID,mucopolysaccharidosis type IVA, Morquio syndrome, mucopolysaccharidosistype IVB, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII,Sly syndrome, mucopolysaccharidosis type IX, multiple sulfatasedeficiency, neuronal ceroid lipofuscinosis, CLN1 Batten disease, CLN2Batten disease, Niemann-Pick disease types A/B, Niemann-Pick diseasetype C1, Niemann-Pick disease type C2, pycnodysostosis, Schindlerdisease types I/II, Gaucher disease and sialic acid storage disease.

A detailed review of the genetic etiology, clinical manifestations, andmolecular biology of the lysosomal storage diseases are detailed inScriver et al., eds., The Metabolic and Molecular Basis of InheritedDisease, 7.sup.th Ed., Vol. II, McGraw Hill, (1995). Thus, the enzymesdeficient in the above diseases are known to those of skill in the art,some of these are exemplified in the Table below:

TABLE 2 Substance Disease Name Enzyme Deficiency Stored Pompe DiseaseAcid-a1, 4- Glycogen α 1-4 linked Glucosidase Oligosaccharides GM1Gangliodsidosis β-Galactosidase GM₁ Gangliosides Tay-Sachs Diseaseβ-Hexosaminidase A GM₂ Ganglioside GM2 Gangliosidosis: GM₂ Activator GM₂Ganglioside AB Variant Protein Sandhoff Disease β-Hexosaminidase GM₂Ganglioside A&B Fabry Disease α-Galactosidase A Globosides GaucherDisease Glucocerebrosidase Glucosylceramide Metachromatic ArylsulfataseA Sulphatides Leukodystrophy Krabbe Disease GalactosylceramidaseGalactocerebroside Niemann Pick, Types Acid Sphingomyelin A & BSphingomyelinase Niemann-Pick, Type Cholesterol Sphingomyelin CEsterification Defect Niemann-Pick, Type Unknown Sphingomyelin D FarberDisease Acid Ceramidase Ceramide Wolman Disease Acid Lipase CholesterylEsters Hurler Syndrome α-L-Iduronidase Heparan & (MPS IH) DermatanSulfates Scheie Syndrome α-L-Iduronidase Heparan & (MPS IS) Dermatan,Sulfates Hurler-Scheie α-L-Iduronidase Heparan & (MPS IH/S) DermatanSulfates Hunter Syndrome Iduronate Sulfatase Heparan & (MPS II) DermatanSulfates Sanfilippo A Heparan N-Sulfatase Heparan (MPS IIIA) SulfateSanfilippo B α-N- Heparan (MPS IIIB) Acetylglucosaminidase SulfateSanfilippo C Acetyl-CoA- Heparan (MPS IIIC) Glucosaminide SulfateAcetyltransferase Sanfilippo D N-Acetylglucosamine- Heparan (MPS IIID)6-Sulfatase Sulfate Morquio B β-Galactosidase Keratan (MPS IVB) SulfateMaroteaux-Lamy Arylsulfatase B Dermatan (MPS VI) Sulfate Sly Syndromeβ-Glucuronidase (MPS VII) α-Mannosidosis α-Mannosidase Mannose/Oligosaccharides β-Mannosidosis β-Mannosidase Mannose/ OligosaccharidesFucosidosis α-L-Fucosidase Fucosyl OligosaccharidesAspartylglucosaminuria N-Aspartyl-β- Aspartylglucosamine GlucosaminidaseAsparagines Sialidosis α-Neuraminidase Sialyloligosaccharides(Mucolipidosis I) Galactosialidosis Lysosomal ProtectiveSialyloligosaccharides (Goldberg Syndrome) Protein Deficiency SchindlerDisease α-N-Acetyl- Galactosaminidase Mucolipidosis II (I-N-Acetylglucosamine- Heparan Sulfate Cell Disease) 1-PhosphotransferaseMucolipidosis III Same as ML II (Pseudo-Hurler Polydystrophy) CystinosisCystine Transport Free Cystine Protein Salla Disease Sialic AcidTransport Free Sialic Acid and Protein Glucuronic Acid Infantile SialicAcid Sialic Acid Transport Free Sialic Acid and Storage Disease ProteinGlucuronic Acid Infantile Neuronal Palmitoyl-Protein Lipofuscins CeroidLipofuscinosis Thioesterase Mucolipidosis IV Unknown Gangliosides &Hyaluronic Acid Prosaposin Saposins A, B, C or D

Inventive methods according to the present invention may be used todeliver various other replacement enzymes. As used herein, replacementenzymes suitable for the present invention may include any enzyme thatcan act to replace at least partial activity of the deficient or missinglysosomal enzyme in a lysosomal storage disease to be treated. In someembodiments, a replacement enzyme is capable of reducing accumulatedsubstance in lysosomes or that can rescue or ameliorate one or morelysosomal storage disease symptoms.

In some embodiments, a suitable replacement enzyme may be any lysosomalenzyme known to be associated with the lysosomal storage disease to betreated. In some embodiments, a suitable replacement enzyme is an enzymeselected from the enzyme listed in Table 2 above.

In some embodiments, a replacement enzyme suitable for the invention mayhave a wild-type or naturally occurring sequence. In some embodiments, areplacement enzyme suitable for the invention may have a modifiedsequence having substantial homology or identify to the wild-type ornaturally-occurring sequence (e.g., having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% sequence identity to the wild-type ornaturally-occurring sequence).

A replacement enzyme suitable for the present invention may be producedby any available means. For example, replacement enzymes may berecombinantly produced by utilizing a host cell system engineered toexpress a replacement enzyme-encoding nucleic acid. Alternatively oradditionally, replacement enzymes may be produced by activatingendogenous genes. Alternatively or additionally, replacement enzymes maybe partially or fully prepared by chemical synthesis. Alternatively oradditionally, replacements enzymes may also be purified from naturalsources.

Where enzymes are recombinantly produced, any expression system can beused. To give but a few examples, known expression systems include, forexample, egg, baculovirus, plant, yeast, or mammalian cells.

In some embodiments, enzymes suitable for the present invention areproduced in mammalian cells. Non-limiting examples of mammalian cellsthat may be used in accordance with the present invention include BALB/cmouse myeloma line (NSO/1, ECACC No: 85110503); human retinoblasts(PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol., 36:59, 1977); human fibrosarcoma cell line (e.g.,HT1080); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells +/−DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77:4216, 1980); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinomacells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

In some embodiments, inventive methods according to the presentinvention are used to deliver replacement enzymes produced from humancells. In some embodiments, inventive methods according to the presentinvention are used to deliver replacement enzymes produced from CHOcells.

In some embodiments, replacement enzymes delivered using a method of theinvention contain a moiety that binds to a receptor on the surface ofbrain cells to facilitate cellular uptake and/or lysosomal targeting.For example, such a receptor may be the cation-independentmannose-6-phosphate receptor (CI-MPR) which binds themannose-6-phosphate (M6P) residues. In addition, the CI-MPR also bindsother proteins including IGF-II. In some embodiments, a replacementenzyme suitable for the present invention contains M6P residues on thesurface of the protein. In some embodiments, a replacement enzymesuitable for the present invention may contain bis-phosphorylatedoligosaccharides which have higher binding affinity to the CI-MPR. Insome embodiments, a suitable enzyme contains up to about an average ofabout at least 20% bis-phosphorylated oligosaccharides per enzyme. Inother embodiments, a suitable enzyme may contain about 10%, 15%, 18%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% bis-phosphorylatedoligosaccharides per enzyme. While such bis-phosphorylatedoligosaccharides may be naturally present on the enzyme, it should benoted that the enzymes may be modified to possess such oligosaccharides.For example, suitable replacement enzymes may be modified by certainenzymes which are capable of catalyzing the transfer ofN-acetylglucosamine-L-phosphate from UDP-GlcNAc to the 6′ position ofα-1,2-linked mannoses on lysosomal enzymes. Methods and compositions forproducing and using such enzymes are described by, for example, Canfieldet al. in U.S. Pat. Nos. 6,537,785, and 6,534,300, each incorporatedherein by reference.

In some embodiments, replacement enzymes for use in the presentinvention may be conjugated or fused to a lysosomal targeting moietythat is capable of binding to a receptor on the surface of brain cells.A suitable lysosomal targeting moiety can be IGF-I, IGF-II, RAP, p97,and variants, homologues or fragments thereof (e.g., including thosepeptide having a sequence at least 70%, 75%, 80%, 85%, 90%, or 95%identical to a wild-type mature human IGF-I, IGF-II, RAP, p97 peptidesequence).

In some embodiments, replacement enzymes suitable for the presentinvention have not been modified to enhance delivery or transport ofsuch agents across the BBB and into the CNS.

In some embodiments, a therapeutic protein includes a targeting moiety(e.g., a lysosome targeting sequence) and/or a membrane-penetratingpeptide. In some embodiments, a targeting sequence and/or amembrane-penetrating peptide is an intrinsic part of the therapeuticmoiety (e.g., via a chemical linkage, via a fusion protein). In someembodiments, a targeting sequence contains a mannose-6-phosphate moiety.In some embodiments, a targeting sequence contains an IGF-I moiety. Insome embodiments, a targeting sequence contains an IGF-II moiety.

Formulations

In some embodiments, desired enzymes are delivered in stableformulations for intrathecal delivery. Certain embodiments of theinvention are based, at least in part, on the discovery that variousformulations disclosed herein facilitate the effective delivery anddistribution of one or more therapeutic agents (e.g., an HNS enzyme) totargeted tissues, cells and/or organelles of the CNS. Among otherthings, formulations described herein are capable of solubilizing highconcentrations of therapeutic agents (e.g., an HNS enzyme) and aresuitable for the delivery of such therapeutic agents to the CNS ofsubjects for the treatment of diseases having a CNS component and/oretiology (e.g., Sanfilippo A Syndrome). The compositions describedherein are further characterized by improved stability and improvedtolerability when administered to the CNS of a subject (e.g.,intrathecally) in need thereof.

Before the present invention, traditional unbuffered isotonic saline andElliott's B solution, which is artificial CSF, were typically used forintrathecal delivery. A comparison depicting the compositions of CSFrelative to Elliott's B solution is included in Table 3 below. As shownin Table 3, the concentration of Elliot's B Solution closely parallelsthat of the CSF. Elliott's B Solution, however contains a very lowbuffer concentration and accordingly may not provide the adequatebuffering capacity needed to stabilize therapeutic agents (e.g.,proteins), especially over extended periods of time (e.g., duringstorage conditions). Furthermore, Elliott's B Solution contains certainsalts which may be incompatible with the formulations intended todeliver some therapeutic agents, and in particular proteins or enzymes.For example, the calcium salts present in Elliott's B Solution arecapable of mediating protein precipitation and thereby reducing thestability of the formulation.

TABLE 3 Na⁺ K⁺ Ca⁺⁺ Mg⁺⁺ HCO3⁻ Cl⁻ Phosphorous Glucose Solution mEq/LmEq/L mEq/L mEq/L mEq/L mEq/L pH mg/L mg/L CSF 117-137 2.3 2.2 2.2 22.9113-127 7.31 1.2-2.1 45-80 Elliott's B 149 2.6 2.7 2.4 22.6 132 6.0-7.52.3 80 Sol'n

Thus, in some embodiments, formulations suitable for CNS deliveryaccording to the present invention are not synthetic or artificial CSF.

In some embodiments, formulations for CNS delivery have been formulatedsuch that they are capable of stabilizing, or alternatively slowing orpreventing the degradation, of a therapeutic agent formulated therewith(e.g., an HNS enzyme). As used herein, the term “stable” refers to theability of the therapeutic agent (e.g., an HNS enzyme) to maintain itstherapeutic efficacy (e.g., all or the majority of its intendedbiological activity and/or physiochemical integrity) over extendedperiods of time. The stability of a therapeutic agent, and thecapability of the pharmaceutical composition to maintain stability ofsuch therapeutic agent, may be assessed over extended periods of time(e.g., preferably for at least 1, 3, 6, 12, 18, 24, 30, 36 months ormore). In the context of a formulation a stable formulation is one inwhich the therapeutic agent therein essentially retains its physicaland/or chemical integrity and biological activity upon storage andduring processes (such as freeze/thaw, mechanical mixing andlyophilization). For protein stability, it can be measure by formationof high molecular weight (HMW) aggregates, loss of enzyme activity,generation of peptide fragments and shift of charge profiles.

Stability of the therapeutic agent is of particular importance.Stability of the therapeutic agent may be further assessed relative tothe biological activity or physiochemical integrity of the therapeuticagent over extended periods of time. For example, stability at a giventime point may be compared against stability at an earlier time point(e.g., upon formulation day 0) or against unformulated therapeutic agentand the results of this comparison expressed as a percentage.Preferably, the pharmaceutical compositions of the present inventionmaintain at least 100%, at least 99%, at least 98%, at least 97% atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55% or at least 50% ofthe therapeutic agent's biological activity or physiochemical integrityover an extended period of time (e.g., as measured over at least about6-12 months, at room temperature or under accelerated storageconditions).

In some embodiments, therapeutic agents (e.g., desired enzymes) aresoluble in formulations of the present invention. The term “soluble” asit relates to the therapeutic agents of the present invention refer tothe ability of such therapeutic agents to form a homogenous solution.Preferably the solubility of the therapeutic agent in the solution intowhich it is administered and by which it is transported to the targetsite of action (e.g., the cells and tissues of the brain) is sufficientto permit the delivery of a therapeutically effective amount of thetherapeutic agent to the targeted site of action. Several factors canimpact the solubility of the therapeutic agents. For example, relevantfactors which may impact protein solubility include ionic strength,amino acid sequence and the presence of other co-solubilizing agents orsalts (e.g., calcium salts.) In some embodiments, the pharmaceuticalcompositions are formulated such that calcium salts are excluded fromsuch compositions.

Suitable formulations, in either aqueous, pre-lyophilized, lyophilizedor reconstituted form, may contain a therapeutic agent of interest atvarious concentrations. In some embodiments, formulations may contain aprotein or therapeutic agent of interest at a concentration in the rangeof about 0.1 mg/ml to 100 mg/ml (e.g., about 0.1 mg/ml to 80 mg/ml,about 0.1 mg/ml to 60 mg/ml, about 0.1 mg/ml to 50 mg/ml, about 0.1mg/ml to 40 mg/ml, about 0.1 mg/ml to 30 mg/ml, about 0.1 mg/ml to 25mg/ml, about 0.1 mg/ml to 20 mg/ml, about 0.1 mg/ml to 60 mg/ml, about0.1 mg/ml to 50 mg/ml, about 0.1 mg/ml to 40 mg/ml, about 0.1 mg/ml to30 mg/ml, about 0.1 mg/ml to 25 mg/ml, about 0.1 mg/ml to 20 mg/ml,about 0.1 mg/ml to 15 mg/ml, about 0.1 mg/ml to 10 mg/ml, about 0.1mg/ml to 5 mg/ml, about 1 mg/ml to 10 mg/ml, about 1 mg/ml to 20 mg/ml,about 1 mg/ml to 40 mg/ml, about 5 mg/ml to 100 mg/ml, about 5 mg/ml to50 mg/ml, or about 5 mg/ml to 25 mg/ml). In some embodiments,formulations according to the invention may contain a therapeutic agentat a concentration of approximately 1 mg/ml, 5 mg/ml, 10 mg/ml, 15mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70mg/ml, 80 mg/ml, 90 mg/ml, or 100 mg/ml.

The formulations of the present invention are characterized by theirtolerability either as aqueous solutions or as reconstituted lyophilizedsolutions. As used herein, the terms “tolerable” and “tolerability”refer to the ability of the pharmaceutical compositions of the presentinvention to not elicit an adverse reaction in the subject to whom suchcomposition is administered, or alternatively not to elicit a seriousadverse reaction in the subject to whom such composition isadministered. In some embodiments, the pharmaceutical compositions ofthe present invention are well tolerated by the subject to whom suchcompositions is administered.

Many therapeutic agents, and in particular the proteins and enzymes ofthe present invention, require controlled pH and specific excipients tomaintain their solubility and stability in the pharmaceuticalcompositions of the present invention. Table 4 below identifies typicalexemplary aspects of protein formulations considered to maintain thesolubility and stability of the protein therapeutic agents of thepresent invention.

TABLE 4 Parameter Typical Range/Type Rationale pH 5 to 7.5 For stabilitySometimes also for solubility Buffer type acetate, succinate, citrate,To maintain optimal pH histidine, phosphate or Tris May also affectstability Buffer 5-50 mM To maintain pH concentration May also stabilizeor add ionic strength Tonicifier NaCl, sugars, mannitol To renderiso-osmotic or isotonic solutions Surfactant Polysorbate 20, Tostabilize against polysorbate 80 interfaces and shear Other Amino acids(e.g. arginine) For enhanced solubility at tens to hundreds of mM orstability

Buffers

The pH of the formulation is an additional factor which is capable ofaltering the solubility of a therapeutic agent (e.g., an enzyme orprotein) in an aqueous formulation or for a pre-lyophilizationformulation. Accordingly the formulations of the present inventionpreferably comprise one or more buffers. In some embodiments the aqueousformulations comprise an amount of buffer sufficient to maintain theoptimal pH of said composition between about 4.0-8.0 (e.g., about 4.0,4.5, 5.0, 5.5, 6.0, 6.2, 6.4, 6.5, 6.6, 6.8, 7.0, 7.5, or 8.0). In someembodiments, the pH of the formulation is between about 5.0-7.5, betweenabout 5.5-7.0, between about 6.0-7.0, between about 5.5-6.0, betweenabout 5.5-6.5, between about 5.0-6.0, between about 5.0-6.5 and betweenabout 6.0-7.5. Suitable buffers include, for example acetate, citrate,histidine, phosphate, succinate, tris(hydroxymethyl)aminomethane(“Tris”) and other organic acids. The buffer concentration and pH rangeof the pharmaceutical compositions of the present invention are factorsin controlling or adjusting the tolerability of the formulation. In someembodiments, a buffering agent is present at a concentration rangingbetween about 1 mM to about 150 mM, or between about 10 mM to about 50mM, or between about 15 mM to about 50 mM, or between about 20 mM toabout 50 mM, or between about 25 mM to about 50 mM. In some embodiments,a suitable buffering agent is present at a concentration ofapproximately 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40mM, 45 mM 50 mM, 75 mM, 100 mM, 125 mM or 150 mM.

Tonicity

In some embodiments, formulations, in either aqueous, pre-lyophilized,lyophilized or reconstituted form, contain an isotonicity agent to keepthe formulations isotonic. Typically, by “isotonic” is meant that theformulation of interest has essentially the same osmotic pressure ashuman blood. Isotonic formulations will generally have an osmoticpressure from about 240 mOsm/kg to about 350 mOsm/kg. Isotonicity can bemeasured using, for example, a vapor pressure or freezing point typeosmometers. Exemplary isotonicity agents include, but are not limitedto, glycine, sorbitol, mannitol, sodium chloride and arginine. In someembodiments, suitable isotonic agents may be present in aqueous and/orpre-lyophilized formulations at a concentration from about 0.01-5%(e.g., 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0,2.5, 3.0, 4.0 or 5.0%) by weight. In some embodiments, formulations forlyophilization contain an isotonicity agent to keep thepre-lyophilization formulations or the reconstituted formulationsisotonic.

While generally isotonic solutions are preferred for parenterallyadministered drugs, the use of isotonic solutions may change solubilityfor some therapeutic agents and in particular some proteins and/orenzymes. Slightly hypertonic solutions (e.g., up to 175 mM sodiumchloride in 5 mM sodium phosphate at pH 7.0) and sugar-containingsolutions (e.g., up to 2% sucrose in 5 mM sodium phosphate at pH 7.0)have been demonstrated to be well tolerated. The most common approvedCNS bolus formulation composition is saline (about 150 mM NaCl inwater).

Stabilizing Agents

In some embodiments, formulations may contain a stabilizing agent, orlyoprotectant, to protect the protein. Typically, a suitable stabilizingagent is a sugar, a non-reducing sugar and/or an amino acid. Exemplarysugars include, but are not limited to, dextran, lactose, mannitol,mannose, sorbitol, raffinose, sucrose and trehalose. Exemplary aminoacids include, but are not limited to, arginine, glycine and methionine.Additional stabilizing agents may include sodium chloride, hydroxyethylstarch and polyvinylpyrolidone. The amount of stabilizing agent in thelyophilized formulation is generally such that the formulation will beisotonic. However, hypertonic reconstituted formulations may also besuitable. In addition, the amount of stabilizing agent must not be toolow such that an unacceptable amount of degradation/aggregation of thetherapeutic agent occurs. Exemplary stabilizing agent concentrations inthe formulation may range from about 1 mM to about 400 mM (e.g., fromabout 30 mM to about 300 mM, and from about 50 mM to about 100 mM), oralternatively, from 0.1% to 15% (e.g., from 1% to 10%, from 5% to 15%,from 5% to 10%) by weight. In some embodiments, the ratio of the massamount of the stabilizing agent and the therapeutic agent is about 1:1.In other embodiments, the ratio of the mass amount of the stabilizingagent and the therapeutic agent can be about 0.1:1, 0.2:1, 0.25:1,0.4:1, 0.5:1, 1:1, 2:1, 2.6:1, 3:1, 4:1, 5:1, 10;1, or 20:1. In someembodiments, suitable for lyophilization, the stabilizing agent is alsoa lyoprotectant.

In some embodiments, liquid formulations suitable for the presentinvention contain amorphous materials. In some embodiments, liquidformulations suitable for the present invention contain a substantialamount of amorphous materials (e.g., sucrose-based formulations). Insome embodiments, liquid formulations suitable for the present inventioncontain partly crystalline/partly amorphous materials.

Bulking Agents

In some embodiments, suitable formulations for lyophilization mayfurther include one or more bulking agents. A “bulking agent” is acompound which adds mass to the lyophilized mixture and contributes tothe physical structure of the lyophilized cake. For example, a bulkingagent may improve the appearance of lyophilized cake (e.g., essentiallyuniform lyophilized cake). Suitable bulking agents include, but are notlimited to, sodium chloride, lactose, mannitol, glycine, sucrose,trehalose, hydroxyethyl starch. Exemplary concentrations of bulkingagents are from about 1% to about 10% (e.g., 1.0%, 1.5%, 2.0%, 2.5%,3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%,9.0%, 9.5%, and 10.0%).

Surfactants

In some embodiments, it is desirable to add a surfactant toformulations. Exemplary surfactants include nonionic surfactants such asPolysorbates (e.g., Polysorbates 20 or 80); poloxamers (e.g., poloxamer188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate;sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroamidopropyl); myristarnidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g., Pluronics, PF68, etc). Typically,the amount of surfactant added is such that it reduces aggregation ofthe protein and minimizes the formation of particulates oreffervescences. For example, a surfactant may be present in aformulation at a concentration from about 0.001-0.5% (e.g., about0.005-0.05%, or 0.005-0.01%). In particular, a surfactant may be presentin a formulation at a concentration of approximately 0.005%, 0.01%,0.02%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%, etc. Alternatively, or inaddition, the surfactant may be added to the lyophilized formulation,pre-lyophilized formulation and/or the reconstituted formulation.

Other pharmaceutically acceptable carriers, excipients or stabilizerssuch as those described in Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980) may be included in the formulation (and/orthe lyophilized formulation and/or the reconstituted formulation)provided that they do not adversely affect the desired characteristicsof the formulation. Acceptable carriers, excipients or stabilizers arenontoxic to recipients at the dosages and concentrations employed andinclude, but are not limited to, additional buffering agents;preservatives; co-solvents; antioxidants including ascorbic acid andmethionine; chelating agents such as EDTA; metal complexes (e.g.,Zn-protein complexes); biodegradable polymers such as polyesters; and/orsalt-forming counterions such as sodium.

Formulations, in either aqueous, pre-lyophilized, lyophilized orreconstituted form, in accordance with the present invention can beassessed based on product quality analysis, reconstitution time (iflyophilized), quality of reconstitution (if lyophilized), high molecularweight, moisture, and glass transition temperature. Typically, proteinquality and product analysis include product degradation rate analysisusing methods including, but not limited to, size exclusion HPLC(SE-HPLC), cation exchange-HPLC (CEX-HPLC), X-ray diffraction (XRD),modulated differential scanning calorimetry (mDSC), reversed phase HPLC(RP-HPLC), multi-angle light scattering (MALS), fluorescence,ultraviolet absorption, nephelometry, capillary electrophoresis (CE),SDS-PAGE, and combinations thereof. In some embodiments, evaluation ofproduct in accordance with the present invention may include a step ofevaluating appearance (either liquid or cake appearance).

Generally, formulations (lyophilized or aqueous) can be stored forextended periods of time at room temperature. Storage temperature maytypically range from 0° C. to 45° C. (e.g., 4° C., 20° C., 25° C., 45°C. etc.). Formulations may be stored for a period of months to a periodof years. Storage time generally will be 24 months, 12 months, 6 months,4.5 months, 3 months, 2 months or 1 month. Formulations can be storeddirectly in the container used for administration, eliminating transfersteps.

Formulations can be stored directly in the lyophilization container (iflyophilized), which may also function as the reconstitution vessel,eliminating transfer steps. Alternatively, lyophilized productformulations may be measured into smaller increments for storage.Storage should generally avoid circumstances that lead to degradation ofthe proteins, including but not limited to exposure to sunlight, UVradiation, other forms of electromagnetic radiation, excessive heat orcold, rapid thermal shock, and mechanical shock.

Lyophilization

Inventive methods in accordance with the present invention can beutilized to lyophilize any materials, in particular, therapeutic agents.Typically, a pre-lyophilization formulation further contains anappropriate choice of excipients or other components such asstabilizers, buffering agents, bulking agents, and surfactants toprevent compound of interest from degradation (e.g., proteinaggregation, deamidation, and/or oxidation) during freeze-drying andstorage. The formulation for lyophilization can include one or moreadditional ingredients including lyoprotectants or stabilizing agents,buffers, bulking agents, isotonicity agents and surfactants.

After the substance of interest and any additional components are mixedtogether, the formulation is lyophilized. Lyophilization generallyincludes three main stages: freezing, primary drying and secondarydrying. Freezing is necessary to convert water to ice or some amorphousformulation components to the crystalline form. Primary drying is theprocess step when ice is removed from the frozen product by directsublimation at low pressure and temperature. Secondary drying is theprocess step when bounded water is removed from the product matrixutilizing the diffusion of residual water to the evaporation surface.Product temperature during secondary drying is normally higher thanduring primary drying. See, Tang X. et al. (2004) “Design offreeze-drying processes for pharmaceuticals: Practical advice,” Pharm.Res., 21:191-200; Nail S. L. et al. (2002) “Fundamentals offreeze-drying,” in Development and manufacture of proteinpharmaceuticals. Nail S. L. editor New York: Kluwer Academic/PlenumPublishers, pp 281-353; Wang et al. (2000) “Lyophilization anddevelopment of solid protein pharmaceuticals,” Int. J. Pharm., 203:1-60;Williams N. A. et al. (1984) “The lyophilization of pharmaceuticals; Aliterature review.” J. Parenteral Sci. Technol., 38:48-59. Generally,any lyophilization process can be used in connection with the presentinvention.

In some embodiments, an annealing step may be introduced during theinitial freezing of the product. The annealing step may reduce theoverall cycle time. Without wishing to be bound by any theories, it iscontemplated that the annealing step can help promote excipientcrystallization and formation of larger ice crystals due tore-crystallization of small crystals formed during supercooling, which,in turn, improves reconstitution. Typically, an annealing step includesan interval or oscillation in the temperature during freezing. Forexample, the freeze temperature may be −40° C., and the annealing stepwill increase the temperature to, for example, −10° C. and maintain thistemperature for a set period of time. The annealing step time may rangefrom 0.5 hours to 8 hours (e.g., 0.5, 1.0 1.5, 2.0, 2.5, 3, 4, 6, and 8hours). The annealing temperature may be between the freezingtemperature and 0° C.

Lyophilization may be performed in a container, such as a tube, a bag, abottle, a tray, a vial (e.g., a glass vial), syringe or any othersuitable containers. The containers may be disposable. Lyophilizationmay also be performed in a large scale or small scale. In someinstances, it may be desirable to lyophilize the protein formulation inthe container in which reconstitution of the protein is to be carriedout in order to avoid a transfer step. The container in this instancemay, for example, be a 3, 4, 5, 10, 20, 50 or 100 cc vial.

Many different freeze-dryers are available for this purpose such as Hullpilot scale dryer (SP Industries, USA), Genesis (SP Industries)laboratory freeze-dryers, or any freeze-dryers capable of controllingthe given lyophilization process parameters. Freeze-drying isaccomplished by freezing the formulation and subsequently subliming icefrom the frozen content at a temperature suitable for primary drying.Initial freezing brings the formulation to a temperature below about−20° C. (e.g., −50° C., −45° C., −40° C., −35° C., −30° C., −25° C.,etc.) in typically not more than about 4 hours (e.g., not more thanabout 3 hours, not more than about 2.5 hours, not more than about 2hours). Under this condition, the product temperature is typically belowthe eutectic point or the collapse temperature of the formulation.Typically, the shelf temperature for the primary drying will range fromabout −30 to 25° C. (provided the product remains below the meltingpoint during primary drying) at a suitable pressure, ranging typicallyfrom about 20 to 250 mTorr. The formulation, size and type of thecontainer holding the sample (e.g., glass vial) and the volume of liquidwill mainly dictate the time required for drying, which can range from afew hours to several days. A secondary drying stage is carried out atabout 0-60° C., depending primarily on the type and size of containerand the type of therapeutic agent employed. Again, volume of liquid willmainly dictate the time required for drying, which can range from a fewhours to several days.

As a general proposition, lyophilization will result in a lyophilizedformulation in which the moisture content thereof is less than about 5%,less than about 4%, less than about 3%, less than about 2%, less thanabout 1%, and less than about 0.5%.

Reconstitution

While the pharmaceutical compositions of the present invention aregenerally in an aqueous form upon administration to a subject, in someembodiments the pharmaceutical compositions of the present invention arelyophilized. Such compositions must be reconstituted by adding one ormore diluents thereto prior to administration to a subject. At thedesired stage, typically at an appropriate time prior to administrationto the patient, the lyophilized formulation may be reconstituted with adiluent such that the protein concentration in the reconstitutedformulation is desirable.

Various diluents may be used in accordance with the present invention.In some embodiments, a suitable diluent for reconstitution is water. Thewater used as the diluent can be treated in a variety of ways includingreverse osmosis, distillation, deionization, filtrations (e.g.,activated carbon, microfiltration, nanofiltration) and combinations ofthese treatment methods. In general, the water should be suitable forinjection including, but not limited to, sterile water or bacteriostaticwater for injection.

Additional exemplary diluents include a pH buffered solution (e.g.,phosphate-buffered saline), sterile saline solution, Elliot's solution,Ringer's solution or dextrose solution. Suitable diluents may optionallycontain a preservative. Exemplary preservatives include aromaticalcohols such as benzyl or phenol alcohol. The amount of preservativeemployed is determined by assessing different preservativeconcentrations for compatibility with the protein and preservativeefficacy testing. For example, if the preservative is an aromaticalcohol (such as benzyl alcohol), it can be present in an amount fromabout 0.1-2.0%, from about 0.5-1.5%, or about 1.0-1.2%.

Diluents suitable for the invention may include a variety of additives,including, but not limited to, pH buffering agents, (e.g. Tris,histidine) salts (e.g., sodium chloride) and other additives (e.g.,sucrose) including those described above (e.g. stabilizing agents,isotonicity agents).

According to the present invention, a lyophilized substance (e.g.,protein) can be reconstituted to a concentration of at least 25 mg/ml(e.g., at least 50 mg/ml, at least 75 mg/ml, at least 100 mg/) and inany ranges therebetween. In some embodiments, a lyophilized substance(e.g., protein) may be reconstituted to a concentration ranging fromabout 1 mg/ml to 100 mg/ml (e.g., from about 1 mg/ml to 50 mg/ml, from 1mg/ml to 100 mg/ml, from about 1 mg/ml to about 5 mg/ml, from about 1mg/ml to about 10 mg/ml, from about 1 mg/ml to about 25 mg/ml, fromabout 1 mg/ml to about 75 mg/ml, from about 10 mg/ml to about 30 mg/ml,from about 10 mg/ml to about 50 mg/ml, from about 10 mg/ml to about 75mg/ml, from about 10 mg/ml to about 100 mg/ml, from about 25 mg/ml toabout 50 mg/ml, from about 25 mg/ml to about 75 mg/ml, from about 25mg/ml to about 100 mg/ml, from about 50 mg/ml to about 75 mg/ml, fromabout 50 mg/ml to about 100 mg/ml). In some embodiments, theconcentration of protein in the reconstituted formulation may be higherthan the concentration in the pre-lyophilization formulation. Highprotein concentrations in the reconstituted formulation are consideredto be particularly useful where subcutaneous or intramuscular deliveryof the reconstituted formulation is intended. In some embodiments, theprotein concentration in the reconstituted formulation may be about 2-50times (e.g., about 2-20, about 2-10 times, or about 2-5 times) of thepre-lyophilized formulation. In some embodiments, the proteinconcentration in the reconstituted formulation may be at least about 2times (e.g., at least about 3, 4, 5, 10, 20, 40 times) of thepre-lyophilized formulation.

Reconstitution according to the present invention may be performed inany container. Exemplary containers suitable for the invention include,but are not limited to, such as tubes, vials, syringes (e.g.,single-chamber or dual-chamber), bags, bottles, and trays. Suitablecontainers may be made of any materials such as glass, plastics, metal.The containers may be disposable or reusable. Reconstitution may also beperformed in a large scale or small scale.

In some instances, it may be desirable to lyophilize the proteinformulation in the container in which reconstitution of the protein isto be carried out in order to avoid a transfer step. The container inthis instance may, for example, be a 3, 4, 5, 10, 20, 50 or 100 cc vial.In some embodiments, a suitable container for lyophilization andreconstitution is a dual chamber syringe (e.g., Lyo-Ject,® (Vetter)syringes). For example, a dual chamber syringe may contain both thelyophilized substance and the diluent, each in a separate chamber,separated by a stopper (see Example 5). To reconstitute, a plunger canbe attached to the stopper at the diluent side and pressed to movediluent into the product chamber so that the diluent can contact thelyophilized substance and reconstitution may take place as describedherein (see Example 5).

The pharmaceutical compositions, formulations and related methods of theinvention are useful for delivering a variety of therapeutic agents tothe CNS of a subject (e.g., intrathecally, intraventricularly orintracisternally) and for the treatment of the associated diseases. Thepharmaceutical compositions of the present invention are particularlyuseful for delivering proteins and enzymes (e.g., enzyme replacementtherapy) to subjects suffering from lysosomal storage disorders. Thelysosomal storage diseases represent a group of relatively rareinherited metabolic disorders that result from defects in lysosomalfunction. The lysosomal diseases are characterized by the accumulationof undigested macromolecules within the lysosomes, which results in anincrease in the size and number of such lysosomes and ultimately incellular dysfunction and clinical abnormalities.

CNS Delivery

It is contemplated that various stable formulations described herein aregenerally suitable for CNS delivery of therapeutic agents. Stableformulations according to the present invention can be used for CNSdelivery via various techniques and routes including, but not limitedto, intraparenchymal, intracerebral, intravetricular cerebral (ICV),intrathecal (e.g., IT-Lumbar, IT-cisterna magna) administrations and anyother techniques and routes for injection directly or indirectly to theCNS and/or CSF.

Intrathecal Delivery

In some embodiments, a replacement enzyme is delivered to the CNS in aformulation described herein. In some embodiments, a replacement enzymeis delivered to the CNS by administering into the cerebrospinal fluid(CSF) of a subject in need of treatment. In some embodiments,intrathecal administration is used to deliver a desired replacementenzyme (e.g., an HNS protein) into the CSF. As used herein, intrathecaladministration (also referred to as intrathecal injection) refers to aninjection into the spinal canal (intrathecal space surrounding thespinal cord). Various techniques may be used including, withoutlimitation, lateral cerebroventricular injection through a burrhole orcistemal or lumbar puncture or the like. Exemplary methods are describedin Lazorthes et al. Advances in Drug Delivery Systems and Applicationsin Neurosurgery, 143-192 and Omaya et al., Cancer Drug Delivery, 1:169-179, the contents of which are incorporated herein by reference.

According to the present invention, an enzyme may be injected at anyregion surrounding the spinal canal. In some embodiments, an enzyme isinjected into the lumbar area or the cisterna magna orintraventricularly into a cerebral ventricle space. As used herein, theterm “lumbar region” or “lumbar area” refers to the area between thethird and fourth lumbar (lower back) vertebrae and, more inclusively,the L2-S1 region of the spine. Typically, intrathecal injection via thelumbar region or lumber area is also referred to as “lumbar IT delivery”or “lumbar IT administration.” The term “cisterna magna” refers to thespace around and below the cerebellum via the opening between the skulland the top of the spine. Typically, intrathecal injection via cisternamagna is also referred to as “cisterna magna delivery.” The term“cerebral ventricle” refers to the cavities in the brain that arecontinuous with the central canal of the spinal cord. Typically,injections via the cerebral ventricle cavities are referred to asintravetricular Cerebral (ICV) delivery.

In some embodiments, “intrathecal administration” or “intrathecaldelivery” according to the present invention refers to lumbar ITadministration or delivery, for example, delivered between the third andfourth lumbar (lower back) vertebrae and, more inclusively, the L2-S1region of the spine. It is contemplated that lumbar IT administration ordelivery distinguishes over cisterna magna delivery in that lumbar ITadministration or delivery according to our invention provides betterand more effective delivery to the distal spinal canal, while cisternamagna delivery, among other things, typically does not deliver well tothe distal spinal canal.

Device for Intrathecal Delivery

Various devices may be used for intrathecal delivery according to thepresent invention. In some embodiments, a device for intrathecaladministration contains a fluid access port (e.g., injectable port); ahollow body (e.g., catheter) having a first flow orifice in fluidcommunication with the fluid access port and a second flow orificeconfigured for insertion into spinal cord; and a securing mechanism forsecuring the insertion of the hollow body in the spinal cord. As anon-limiting example shown in FIG. 36, a suitable securing mechanismcontains one or more nobs mounted on the surface of the hollow body anda sutured ring adjustable over the one or more nobs to prevent thehollow body (e.g., catheter) from slipping out of the spinal cord. Invarious embodiments, the fluid access port comprises a reservoir. Insome embodiments, the fluid access port comprises a mechanical pump(e.g., an infusion pump). In some embodiments, an implanted catheter isconnected to either a reservoir (e.g., for bolus delivery), or aninfusion pump. The fluid access port may be implanted or external

In some embodiments, intrathecal administration may be performed byeither lumbar puncture (i.e., slow bolus) or via a port-catheterdelivery system (i.e., infusion or bolus). In some embodiments, thecatheter is inserted between the laminae of the lumbar vertebrae and thetip is threaded up the thecal space to the desired level (generallyL3-L4) (FIG. 37A-C).

Relative to intravenous administration, a single dose volume suitablefor intrathecal administration is typically small. Typically,intrathecal delivery according to the present invention maintains thebalance of the composition of the CSF as well as the intracranialpressure of the subject. In some embodiments, intrathecal delivery isperformed absent the corresponding removal of CSF from a subject. Insome embodiments, a suitable single dose volume may be e.g., less thanabout 10 ml, 8 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1.5 ml, 1 ml, or 0.5ml. In some embodiments, a suitable single dose volume may be about0.5-5 ml, 0.5-4 ml, 0.5-3 ml, 0.5-2 ml, 0.5-1 ml, 1-3 ml, 1-5 ml, 1.5-3ml, 1-4 ml, or 0.5-1.5 ml. In some embodiments, intrathecal deliveryaccording to the present invention involves a step of removing a desiredamount of CSF first. In some embodiments, less than about 10 ml (e.g.,less than about 9 ml, 8 ml, 7 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml) ofCSF is first removed before IT administration. In those cases, asuitable single dose volume may be e.g., more than about 3 ml, 4 ml, 5ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml.

Various other devices may be used to effect intrathecal administrationof a therapeutic composition. For example, formulations containingdesired enzymes may be given using an Ommaya reservoir which is incommon use for intrathecally administering drugs for meningealcarcinomatosis (Lancet 2: 983-84, 1963). More specifically, in thismethod, a ventricular tube is inserted through a hole formed in theanterior horn and is connected to an Ommaya reservoir installed underthe scalp, and the reservoir is subcutaneously punctured tointrathecally deliver the particular enzyme being replaced, which isinjected into the reservoir. Other devices for intrathecaladministration of therapeutic compositions or formulations to anindividual are described in U.S. Pat. No. 6,217,552, incorporated hereinby reference. Alternatively, the drug may be intrathecally given, forexample, by a single injection, or continuous infusion. It should beunderstood that the dosage treatment may be in the form of a single doseadministration or multiple doses.

For injection, formulations of the invention can be formulated in liquidsolutions. In addition, the enzyme may be formulated in solid form andre-dissolved or suspended immediately prior to use. Lyophilized formsare also included. The injection can be, for example, in the form of abolus injection or continuous infusion (e.g., using infusion pumps) ofthe enzyme.

In one embodiment of the invention, the enzyme is administered bylateral cerebro ventricular injection into the brain of a subject. Theinjection can be made, for example, through a burr hole made in thesubject's skull. In another embodiment, the enzyme and/or otherpharmaceutical formulation is administered through a surgically insertedshunt into the cerebral ventricle of a subject. For example, theinjection can be made into the lateral ventricles, which are larger. Insome embodiments, injection into the third and fourth smaller ventriclescan also be made.

In yet another embodiment, the pharmaceutical compositions used in thepresent invention are administered by injection into the cisterna magna,or lumbar area of a subject.

In another embodiment of the method of the invention, thepharmaceutically acceptable formulation provides sustained delivery,e.g., “slow release” of the enzyme or other pharmaceutical compositionused in the present invention, to a subject for at least one, two,three, four weeks or longer periods of time after the pharmaceuticallyacceptable formulation is administered to the subject.

As used herein, the term “sustained delivery” refers to continualdelivery of a pharmaceutical formulation of the invention in vivo over aperiod of time following administration, preferably at least severaldays, a week or several weeks. Sustained delivery of the composition canbe demonstrated by, for example, the continued therapeutic effect of theenzyme over time (e.g., sustained delivery of the enzyme can bedemonstrated by continued reduced amount of storage granules in thesubject). Alternatively, sustained delivery of the enzyme may bedemonstrated by detecting the presence of the enzyme in vivo over time.

Delivery to Target Tissues

As discussed above, one of the surprising and important features of thepresent invention is that therapeutic agents, in particular, replacementenzymes administered using inventive methods and compositions of thepresent invention are able to effectively and extensively diffuse acrossthe brain surface and penetrate various layers or regions of the brain,including deep brain regions. In addition, inventive methods andcompositions of the present invention effectively deliver therapeuticagents (e.g., an HNS enzyme) to various tissues, neurons or cells ofspinal cord, including the lumbar region, which is hard to target byexisting CNS delivery methods such as ICV injection. Furthermore,inventive methods and compositions of the present invention deliversufficient amount of therapeutic agents (e.g., an HNS enzyme) to bloodstream and various peripheral organs and tissues.

Thus, in some embodiments, a therapeutic protein (e.g., an HNS enzyme)is delivered to the central nervous system of a subject. In someembodiments, a therapeutic protein (e.g., an HNS enzyme) is delivered toone or more of target tissues of brain, spinal cord, and/or peripheralorgans. As used herein, the term “target tissues” refers to any tissuethat is affected by the lysosomal storage disease to be treated or anytissue in which the deficient lysosomal enzyme is normally expressed. Insome embodiments, target tissues include those tissues in which there isa detectable or abnormally high amount of enzyme substrate, for examplestored in the cellular lysosomes of the tissue, in patients sufferingfrom or susceptible to the lysosomal storage disease. In someembodiments, target tissues include those tissues that displaydisease-associated pathology, symptom, or feature. In some embodiments,target tissues include those tissues in which the deficient lysosomalenzyme is normally expressed at an elevated level. As used herein, atarget tissue may be a brain target tissue, a spinal cord target tissueand/or a peripheral target tissue. Exemplary target tissues aredescribed in detail below.

Brain Target Tissues

In general, the brain can be divided into different regions, layers andtissues. For example, meningeal tissue is a system of membranes whichenvelops the central nervous system, including the brain. The meningescontain three layers, including dura matter, arachnoid matter, and piamatter. In general, the primary function of the meninges and of thecerebrospinal fluid is to protect the central nervous system. In someembodiments, a therapeutic protein in accordance with the presentinvention is delivered to one or more layers of the meninges.

The brain has three primary subdivisions, including the cerebrum,cerebellum, and brain stem. The cerebral hemispheres, which are situatedabove most other brain structures and are covered with a cortical layer.Underneath the cerebrum lies the brainstem, which resembles a stalk onwhich the cerebrum is attached. At the rear of the brain, beneath thecerebrum and behind the brainstem, is the cerebellum.

The diencephalon, which is located near the midline of the brain andabove the mesencephalon, contains the thalamus, metathalamus,hypothalamus, epithalamus, prethalamus, and pretectum. Themesencephalon, also called the midbrain, contains the tectum,tegumentum, ventricular mesocoelia, and cerebral peduncels, the rednucleus, and the cranial nerve III nucleus. The mesencephalon isassociated with vision, hearing, motor control, sleep/wake, alertness,and temperature regulation.

Regions of tissues of the central nervous system, including the brain,can be characterized based on the depth of the tissues. For example, CNS(e.g., brain) tissues can be characterized as surface or shallowtissues, mid-depth tissues, and/or deep tissues.

According to the present invention, a therapeutic protein (e.g., areplacement enzyme) may be delivered to any appropriate brain targettissue(s) associated with a particular disease to be treated in asubject. In some embodiments, a therapeutic protein (e.g., a replacementenzyme) in accordance with the present invention is delivered to surfaceor shallow brain target tissue. In some embodiments, a therapeuticprotein in accordance with the present invention is delivered tomid-depth brain target tissue. In some embodiments, a therapeuticprotein in accordance with the present invention is delivered to deepbrain target tissue. In some embodiments, a therapeutic protein inaccordance with the present invention is delivered to a combination ofsurface or shallow brain target tissue, mid-depth brain target tissue,and/or deep brain target tissue. In some embodiments, a therapeuticprotein in accordance with the present invention is delivered to a deepbrain tissue at least 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm or morebelow (or internal to) the external surface of the brain.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more surface or shallow tissues of cerebrum. In some embodiments,the targeted surface or shallow tissues of the cerebrum are locatedwithin 4 mm from the surface of the cerebrum. In some embodiments, thetargeted surface or shallow tissues of the cerebrum are selected frompia mater tissues, cerebral cortical ribbon tissues, hippocampus,Virchow Robin space, blood vessels within the VR space, the hippocampus,portions of the hypothalamus on the inferior surface of the brain, theoptic nerves and tracts, the olfactory bulb and projections, andcombinations thereof.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more deep tissues of the cerebrum. In some embodiments, thetargeted surface or shallow tissues of the cerebrum are located 4 mm(e.g., 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm) below (or internal to)the surface of the cerebrum. In some embodiments, targeted deep tissuesof the cerebrum include the cerebral cortical ribbon. In someembodiments, targeted deep tissues of the cerebrum include one or moreof the diencephalon (e.g., the hypothalamus, thalamus, prethalamus,subthalamus, etc.), metencephalon, lentiform nuclei, the basal ganglia,caudate, putamen, amygdala, globus pallidus, and combinations thereof.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more tissues of the cerebellum. In certain embodiments, thetargeted one or more tissues of the cerebellum are selected from thegroup consisting of tissues of the molecular layer, tissues of thePurkinje cell layer, tissues of the Granular cell layer, cerebellarpeduncles, and combination thereof. In some embodiments, therapeuticagents (e.g., enzymes) are delivered to one or more deep tissues of thecerebellum including, but not limited to, tissues of the Purkinje celllayer, tissues of the Granular cell layer, deep cerebellar white mattertissue (e.g., deep relative to the Granular cell layer), and deepcerebellar nuclei tissue.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more tissues of the brainstem. In some embodiments, the targetedone or more tissues of the brainstem include brain stem white mattertissue and/or brain stem nuclei tissue.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered tovarious brain tissues including, but not limited to, gray matter, whitematter, periventricular areas, pia-arachnoid, meninges, neocortex,cerebellum, deep tissues in cerebral cortex, molecular layer,caudate/putamen region, midbrain, deep regions of the pons or medulla,and combinations thereof.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered tovarious cells in the brain including, but not limited to, neurons, glialcells, perivascular cells and/or meningeal cells. In some embodiments, atherapeutic protein is delivered to oligodendrocytes of deep whitematter.

Spinal Cord

In general, regions or tissues of the spinal cord can be characterizedbased on the depth of the tissues. For example, spinal cord tissues canbe characterized as surface or shallow tissues, mid-depth tissues,and/or deep tissues.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more surface or shallow tissues of the spinal cord. In someembodiments, a targeted surface or shallow tissue of the spinal cord islocated within 4 mm from the surface of the spinal cord. In someembodiments, a targeted surface or shallow tissue of the spinal cordcontains pia matter and/or the tracts of white matter.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toone or more deep tissues of the spinal cord. In some embodiments, atargeted deep tissue of the spinal cord is located internal to 4 mm fromthe surface of the spinal cord. In some embodiments, a targeted deeptissue of the spinal cord contains spinal cord grey matter and/orependymal cells.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered toneurons of the spinal cord.

Peripheral Target Tissues

As used herein, peripheral organs or tissues refer to any organs ortissues that are not part of the central nervous system (CNS).Peripheral target tissues may include, but are not limited to, bloodsystem, liver, kidney, heart, endothelium, bone marrow and bone marrowderived cells, spleen, lung, lymph node, bone, cartilage, ovary andtestis. In some embodiments, a therapeutic protein (e.g., a replacementenzyme) in accordance with the present invention is delivered to one ormore of the peripheral target tissues.

Biodistribution and Bioavailability

In various embodiments, once delivered to the target tissue, atherapeutic agent (e.g., an HNS enzyme) is localized intracellularly.For example, a therapeutic agent (e.g., enzyme) may be localized toexons, axons, lysosomes, mitochondria or vacuoles of a target cell(e.g., neurons such as Purkinje cells). For example, in some embodimentsintrathecally-administered enzymes demonstrate translocation dynamicssuch that the enzyme moves within the perivascular space (e.g., bypulsation-assisted convective mechanisms). In addition, active axonaltransport mechanisms relating to the association of the administeredprotein or enzyme with neurofilaments may also contribute to orotherwise facilitate the distribution of intrathecally-administeredproteins or enzymes into the deeper tissues of the central nervoussystem.

In some embodiments, a therapeutic agent (e.g., an HNS enzyme) deliveredaccording to the present invention may achieve therapeutically orclinically effective levels or activities in various targets tissuesdescribed herein. As used herein, a therapeutically or clinicallyeffective level or activity is a level or activity sufficient to confera therapeutic effect in a target tissue. The therapeutic effect may beobjective (i.e., measurable by some test or marker) or subjective (i.e.,subject gives an indication of or feels an effect). For example, atherapeutically or clinically effective level or activity may be anenzymatic level or activity that is sufficient to ameliorate symptomsassociated with the disease in the target tissue (e.g., GAG storage).

In some embodiments, a therapeutic agent (e.g., a replacement enzyme)delivered according to the present invention may achieve an enzymaticlevel or activity that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95% of the normal level or activity of the correspondinglysosomal enzyme in the target tissue. In some embodiments, atherapeutic agent (e.g., a replacement enzyme) delivered according tothe present invention may achieve an enzymatic level or activity that isincreased by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold or 10-fold as compared to a control (e.g.,endogenous levels or activities without the treatment). In someembodiments, a therapeutic agent (e.g., a replacement enzyme) deliveredaccording to the present invention may achieve an increased enzymaticlevel or activity at least approximately 10 nmol/hr/mg, 20 nmol/hr/mg,40 nmol/hr/mg, 50 nmol/hr/mg, 60 nmol/hr/mg, 70 nmol/hr/mg, 80nmol/hr/mg, 90 nmol/hr/mg, 100 nmol/hr/mg, 150 nmol/hr/mg, 200nmol/hr/mg, 250 nmol/hr/mg, 300 nmol/hr/mg, 350 nmol/hr/mg, 400nmol/hr/mg, 450 nmol/hr/mg, 500 nmol/hr/mg, 550 nmol/hr/mg or 600nmol/hr/mg in a target tissue.

In some embodiments, inventive methods according to the presentinvention are particularly useful for targeting the lumbar region. Insome embodiments, a therapeutic agent (e.g., a replacement enzyme)delivered according to the present invention may achieve an increasedenzymatic level or activity in the lumbar region of at leastapproximately 500 nmol/hr/mg, 600 nmol/hr/mg, 700 nmol/hr/mg, 800nmol/hr/mg, 900 nmol/hr/mg, 1000 nmol/hr/mg, 1500 nmol/hr/mg, 2000nmol/hr/mg, 3000 nmol/hr/mg, 4000 nmol/hr/mg, 5000 nmol/hr/mg, 6000nmol/hr/mg, 7000 nmol/hr/mg, 8000 nmol/hr/mg, 9000 nmol/hr/mg, or 10,000nmol/hr/mg.

In general, therapeutic agents (e.g., replacement enzymes) deliveredaccording to the present invention have sufficiently long half time inCSF and target tissues of the brain, spinal cord, and peripheral organs.In some embodiments, a therapeutic agent (e.g., a replacement enzyme)delivered according to the present invention may have a half-life of atleast approximately 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10hours, 12 hours, 16 hours, 18 hours, 20 hours, 25 hours, 30 hours, 35hours, 40 hours, up to 3 days, up to 7 days, up to 14 days, up to 21days or up to a month. In some embodiments, In some embodiments, atherapeutic agent (e.g., a replacement enzyme) delivered according tothe present invention may retain detectable level or activity in CSF orbloodstream after 12 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90hours, 96 hours, 102 hours, or a week following administration.Detectable level or activity may be determined using various methodsknown in the art.

In certain embodiments, a therapeutic agent (e.g., a replacement enzyme)delivered according to the present invention achieves a concentration ofat least 30 μg/ml in the CNS tissues and cells of the subject followingadministration (e.g., one week, 3 days, 48 hours, 36 hours, 24 hours, 18hours, 12 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30minutes, or less, following intrathecal administration of thepharmaceutical composition to the subject). In certain embodiments, atherapeutic agent (e.g., a replacement enzyme) delivered according tothe present invention achieves a concentration of at least 20 μg/ml, atleast 15 μg/ml, at least 10 μg/ml, at least 7.5 μg/ml, at least 5 μg/ml,at least 2.5 μg/ml, at least 1.0 μg/ml or at least 0.5 μg/ml in thetargeted tissues or cells of the subject (e.g., brain tissues orneurons) following administration to such subject (e.g., one week, 3days, 48 hours, 36 hours, 24 hours, 18 hours, 12 hours, 8 hours, 6hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, or less followingintrathecal administration of such pharmaceutical compositions to thesubject).

Treatment of Sanfilippo a Syndrome and Other Lysosomal Storage Diseases

The lysosomal storage diseases represent a group of relatively rareinherited metabolic disorders that result from defects in lysosomalfunction. The lysosomal diseases are characterized by the accumulationof undigested macromolecules, including those enzyme substrates, withinthe lysosomes (see Table 1), which results in an increase in the sizeand number of such lysosomes and ultimately in cellular dysfunction andclinical abnormalities.

Inventive methods described herein can advantageously facilitate thedelivery of one or more therapeutic agents (e.g., one or morereplacement enzymes) to targeted organelles. For example, becauselysosomal storage disorders such as Sanfilippo syndrome Type A arecharacterized by an accumulation of glycosaminoglycans (GAG) in thelysosomes of affected cells, the lysosomes represent an desired targetorganelle for the treatment of the lysosomal storage disorders.

Inventive methods and compositions of the present invention areparticularly useful for treating those diseases having a CNS etiology orcomponent. Lysosomal storage diseases having a CNS etiology orcomponent, include for example and without limitation Sanfilipposyndrome Type A, Sanfilippo syndrome type B, Hunter syndrome,metachromatic leukodystrophy and globoid cell leukodystrophy. Prior tothe present invention, traditional therapies are limited in that theyare administered to subjects intravenously, and are generally onlyeffective in treating the somatic symptoms of the underlying enzymedeficiency. The compositions and methods of the present invention mayadvantageously be administered directly into the CNS of a subjectsuffering from a disease having such a CNS etiology thereby achieving atherapeutic concentration within the affected cells and tissues of theCNS (e.g., the brain), thus overcoming the limitations associated withtraditional systemic administration of such therapeutic agents.

In some embodiments, inventive methods and compositions of the inventionare useful for treating both the neurologic and the somatic sequelae orsymptoms of lysosomal storage disorders. For example, some embodimentsof the invention relate to compositions and methods of delivering one ormore therapeutic agents to the CNS of a subject (e.g., intrathecally,intraventricularly or intracisternally) for the treatment of the CNS orneurologic sequelae and manifestations of a lysosomal storage disease,while also treating the systemic or somatic manifestations of thatlysosomal storage disease. For example, some compositions of the presentinvention may be administered to a subject intrathecally, therebydelivering one or more therapeutic agents to the CNS of the subject andtreating the neurological sequelae, coupled with the intravenousadministration of one or more therapeutic agents to deliver suchtherapeutic agents to both the cells and tissues of the systemiccirculation (e.g., cells and tissues of heart, lungs, liver, kidney orlymph nodes) to thereby treat the somatic sequelae. For example, asubject having or otherwise affected by a lysosomal storage disease(e.g., Sanfilippo Syndrome Type A) may be administered a pharmaceuticalcomposition comprising one or more therapeutic agents (e.g., HNS)intrathecally at least once per week, biweekly, monthly, bimonthly ormore to treat the neurologic sequelae, while a different therapeuticagent is administered to the subject intravenously on a more frequentbasis (e.g., once per day, every other day, three times a week orweekly) to treat the systemic or somatic manifestations of the disease.

Sanfilippo syndrome, or mucopolysaccharidosis III (MPS III), is a raregenetic disorder characterized by the deficiency of enzymes involved inthe degradation of glycosaminoglycans (GAG). In the absence of enzyme,partially degraded GAG molecules cannot be cleared from the body andaccumulate in lysosomes of various tissues, resulting in progressivewidespread somatic dysfunction (Neufeld and Muenzer, 2001).

Four distinct forms of MPS III, designated MPS IIIA, B, C, and D, havebeen identified. Each represents a deficiency in one of four enzymesinvolved in the degradation of the GAG heparan sulfate. All formsinclude varying degrees of the same clinical symptoms, including coarsefacial features, hepatosplenomegaly, corneal clouding and skeletaldeformities. Most notably, however, is the severe and progressive lossof cognitive ability, which is tied not only to the accumulation ofheparan sulfate in neurons, but also the subsequent elevation of thegangliosides GM2, GM3 and GD2 caused by primary GAG accumulation(Walkley 1998).

Mucopolysaccharidosis type IIIA (MPS IIIA; Sanfilippo Syndrome Type A)is the most severe form of Sanfilippo syndrome and affects approximately1 in 100,000 people worldwide. Sanfilippo Syndrome Type A (SanA) ischaracterized by a deficiency of the enzyme heparan N-sulfatase (HNS),an exosulfatase involved in the lysosomal catabolism ofglycosaminoglycan (GAG) heparan sulfate (Neufeld E F, et al. TheMetabolic and Molecular Bases of Inherited Disease (2001) pp.3421-3452). In the absence of this enzyme, GAG heparan sulfateaccumulates in lysosomes of neurons and glial cells, with lesseraccumulation outside the brain.

A defining clinical feature of this disorder is central nervous system(CNS) degeneration, which results in loss of, or failure to attain,major developmental milestones. The progressive cognitive declineculminates in dementia and premature mortality. The disease typicallymanifests itself in young children, and the lifespan of an affectedindividual generally does not extend beyond late teens to earlytwenties.

Compositions and methods of the present invention may be used toeffectively treat individuals suffering from or susceptible toSanfilippo Syndrome Type A. The terms, “treat” or “treatment,” as usedherein, refers to amelioration of one or more symptoms associated withthe disease, prevention or delay of the onset or progression of one ormore symptoms of the disease, and/or lessening of the severity orfrequency of one or more symptoms of the disease.

In some embodiments, treatment refers to partially or completealleviation, amelioration, relief, inhibition, delaying onset, reducingseverity and/or incidence of neurological impairment in a SanA patient.As used herein, the term “neurological impairment” includes varioussymptoms associated with impairment of the central nervous system (e.g.,the brain and spinal cord). Symptoms of neurological impairment mayinclude, for example, developmental delay, progressive cognitiveimpairment, hearing loss, impaired speech development, deficits in motorskills, hyperactivity, aggressiveness and/or sleep disturbances, amongothers.

In some embodiments, treatment refers to decreased lysosomal storage(e.g., of GAG) in various tissues. In some embodiments, treatment refersto decreased lysosomal storage in brain target tissues, spinal cordneurons, and/or peripheral target tissues. In certain embodiments,lysosomal storage is decreased by about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% ormore as compared to a control. In some embodiments, lysosomal storage isdecreased by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold or 10-fold as compared to a control. In someembodiments, lysosomal storage is measured by the presence of lysosomalstorage granules (e.g., zebra-striped morphology).

In some embodiments, treatment refers to reduced vacuolization inneurons (e.g., neurons containing Purkinje cells). In certainembodiments, vacuolization in neurons is decreased by about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100% or more as compared to a control. In someembodiments, vacuolization is decreased by at least 1-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold ascompared to a control.

In some embodiments, treatment refers to increased HNS enzyme activityin various tissues. In some embodiments, treatment refers to increasedHNS enzyme activity in brain target tissues, spinal cord neurons and/orperipheral target tissues. In some embodiments, HNS enzyme activity isincreased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%,600%, 700%, 800%, 900% 1000% or more as compared to a control. In someembodiments, HNS enzyme activity is increased by at least 1-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or10-fold as compared to a control. In some embodiments, increased HNSenzymatic activity is at least approximately 10 nmol/hr/mg, 20nmol/hr/mg, 40 nmol/hr/mg, 50 nmol/hr/mg, 60 nmol/hr/mg, 70 nmol/hr/mg,80 nmol/hr/mg, 90 nmol/hr/mg, 100 nmol/hr/mg, 150 nmol/hr/mg, 200nmol/hr/mg, 250 nmol/hr/mg, 300 nmol/hr/mg, 350 nmol/hr/mg, 400nmol/hr/mg, 450 nmol/hr/mg, 500 nmol/hr/mg, 550 nmol/hr/mg, 600nmol/hr/mg or more. In some embodiments, HNS enzymatic activity isincreased in the lumbar region. In some embodiments, increased HNSenzymatic activity in the lumbar region is at least approximately 2000nmol/hr/mg, 3000 nmol/hr/mg, 4000 nmol/hr/mg, 5000 nmol/hr/mg, 6000nmol/hr/mg, 7000 nmol/hr/mg, 8000 nmol/hr/mg, 9000 nmol/hr/mg, 10,000nmol/hr/mg, or more.

In some embodiments, treatment refers to decreased progression of lossof cognitive ability. In certain embodiments, progression of loss ofcognitive ability is decreased by about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% ormore as compared to a control. In some embodiments, treatment refers todecreased developmental delay. In certain embodiments, developmentaldelay is decreased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more ascompared to a control.

In some embodiments, treatment refers to increased survival (e.g.survival time). For example, treatment can result in an increased lifeexpectancy of a patient. In some embodiments, treatment according to thepresent invention results in an increased life expectancy of a patientby more than about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, about 100%, about 105%, about 110%, about 115%, about 120%, about125%, about 130%, about 135%, about 140%, about 145%, about 150%, about155%, about 160%, about 165%, about 170%, about 175%, about 180%, about185%, about 190%, about 195%, about 200% or more, as compared to theaverage life expectancy of one or more control individuals with similardisease without treatment. In some embodiments, treatment according tothe present invention results in an increased life expectancy of apatient by more than about 6 month, about 7 months, about 8 months,about 9 months, about 10 months, about 11 months, about 12 months, about2 years, about 3 years, about 4 years, about 5 years, about 6 years,about 7 years, about 8 years, about 9 years, about 10 years or more, ascompared to the average life expectancy of one or more controlindividuals with similar disease without treatment. In some embodiments,treatment according to the present invention results in long termsurvival of a patient. As used herein, the term “long term survival”refers to a survival time or life expectancy longer than about 40 years,45 years, 50 years, 55 years, 60 years, or longer.

The terms, “improve,” “increase” or “reduce,” as used herein, indicatevalues that are relative to a control. In some embodiments, a suitablecontrol is a baseline measurement, such as a measurement in the sameindividual prior to initiation of the treatment described herein, or ameasurement in a control individual (or multiple control individuals) inthe absence of the treatment described herein. A “control individual” isan individual afflicted with Sanfilippo Syndrome Type A, who is aboutthe same age and/or gender as the individual being treated (to ensurethat the stages of the disease in the treated individual and the controlindividual(s) are comparable).

The individual (also referred to as “patient” or “subject”) beingtreated is an individual (fetus, infant, child, adolescent, or adulthuman) having Sanfilippo Syndrome Type A or having the potential todevelop Sanfilippo Syndrome Type A. The individual can have residualendogenous HNS expression and/or activity, or no measurable activity.For example, the individual having Sanfilippo Syndrome Type A may haveHNS expression levels that are less than about 30-50%, less than about25-30%, less than about 20-25%, less than about 15-20%, less than about10-15%, less than about 5-10%, less than about 0.1-5% of normal HNSexpression levels.

In some embodiments, the individual is an individual who has beenrecently diagnosed with the disease. Typically, early treatment(treatment commencing as soon as possible after diagnosis) is importantto minimize the effects of the disease and to maximize the benefits oftreatment.

Immune Tolerance

Generally, intrathecal administration of a therapeutic agent (e.g., areplacement enzyme) according to the present invention does not resultin severe adverse effects in the subject. As used herein, severe adverseeffects induce, but are not limited to, substantial immune response,toxicity, or death. As used herein, the term “substantial immuneresponse” refers to severe or serious immune responses, such as adaptiveT-cell immune responses.

Thus, in many embodiments, inventive methods according to the presentinvention do not involve concurrent immunosuppressant therapy (i.e., anyimmunosuppressant therapy used as pre-treatment/pre-conditioning or inparallel to the method). In some embodiments, inventive methodsaccording to the present invention do not involve an immune toleranceinduction in the subject being treated. In some embodiments, inventivemethods according to the present invention do not involve apre-treatment or preconditioning of the subject using T-cellimmunosuppressive agent.

In some embodiments, intrathecal administration of therapeutic agentscan mount an immune response against these agents. Thus, in someembodiments, it may be useful to render the subject receiving thereplacement enzyme tolerant to the enzyme replacement therapy. Immunetolerance may be induced using various methods known in the art. Forexample, an initial 30-60 day regimen of a T-cell immunosuppressiveagent such as cyclosporin A (CsA) and an antiproliferative agent, suchas, azathioprine (Aza), combined with weekly intrathecal infusions oflow doses of a desired replacement enzyme may be used.

Any immunosuppressant agent known to the skilled artisan may be employedtogether with a combination therapy of the invention. Suchimmunosuppressant agents include but are not limited to cyclosporine,FK506, rapamycin, CTLA4-Ig, and anti-TNF agents such as etanercept (seee.g. Moder, 2000, Ann. Allergy Asthma Immunol. 84, 280-284; Nevins,2000, Curr. Opin. Pediatr. 12, 146-150; Kurlberg et al., 2000, Scand. J.Immunol. 51, 224-230; Ideguchi et al., 2000, Neuroscience 95, 217-226;Potter et al., 1999, Ann. N.Y. Acad. Sci. 875, 159-174; Slavik et al.,1999, Immunol. Res. 19, 1-24; Gaziev et al., 1999, Bone MarrowTransplant. 25, 689-696; Henry, 1999, Clin. Transplant. 13, 209-220;Gummert et al., 1999, J. Am. Soc. Nephrol. 10, 1366-1380; Qi et al.,2000, Transplantation 69, 1275-1283). The anti-IL2 receptor(.alpha.-subunit) antibody daclizumab (e.g. Zenapax™), which has beendemonstrated effective in transplant patients, can also be used as animmunosuppressant agent (see e.g. Wiseman et al., 1999, Drugs 58,1029-1042; Beniaminovitz et al., 2000, N. Engl J. Med. 342, 613-619;Ponticelli et al., 1999, Drugs R. D. 1, 55-60; Berard et al., 1999,Pharmacotherapy 19, 1127-1137; Eckhoff et al., 2000, Transplantation 69,1867-1872; Ekberg et al., 2000, Transpl. Int. 13, 151-159). Additionalimmunosuppressant agents include but are not limited to anti-CD2 (Brancoet al., 1999, Transplantation 68, 1588-1596; Przepiorka et al., 1998,Blood 92, 4066-4071), anti-CD4 (Marinova-Mutafchieva et al., 2000,Arthritis Rheum. 43, 638-644; Fishwild et al., 1999, Clin. Immunol. 92,138-152), and anti-CD40 ligand (Hong et al., 2000, Semin. Nephrol. 20,108-125; Chirmule et al., 2000, J. Virol. 74, 3345-3352; Ito et al.,2000, J. Immunol. 164, 1230-1235).

Administration

Inventive methods of the present invention contemplate single as well asmultiple administrations of a therapeutically effective amount of thetherapeutic agents (e.g., replacement enzymes) described herein.Therapeutic agents (e.g., replacement enzymes) can be administered atregular intervals, depending on the nature, severity and extent of thesubject's condition (e.g., a lysosomal storage disease). In someembodiments, a therapeutically effective amount of the therapeuticagents (e.g., replacement enzymes) of the present invention may beadministered intrathecally periodically at regular intervals (e.g., onceevery year, once every six months, once every five months, once everythree months, bimonthly (once every two months), monthly (once everymonth), biweekly (once every two weeks), weekly).

In some embodiments, intrathecal administration may be used inconjunction with other routes of administration (e.g., intravenous,subcutaneously, intramuscularly, parenterally, transdermally, ortransmucosally (e.g., orally or nasally)). In some embodiments, thoseother routes of administration (e.g., intravenous administration) may beperformed no more frequent than biweekly, monthly, once every twomonths, once every three months, once every four months, once every fivemonths, once every six months, annually administration.

As used herein, the term “therapeutically effective amount” is largelydetermined base on the total amount of the therapeutic agent containedin the pharmaceutical compositions of the present invention. Generally,a therapeutically effective amount is sufficient to achieve a meaningfulbenefit to the subject (e.g., treating, modulating, curing, preventingand/or ameliorating the underlying disease or condition). For example, atherapeutically effective amount may be an amount sufficient to achievea desired therapeutic and/or prophylactic effect, such as an amountsufficient to modulate lysosomal enzyme receptors or their activity tothereby treat such lysosomal storage disease or the symptoms thereof(e.g., a reduction in or elimination of the presence or incidence of“zebra bodies” or cellular vacuolization following the administration ofthe compositions of the present invention to a subject). Generally, theamount of a therapeutic agent (e.g., a recombinant lysosomal enzyme)administered to a subject in need thereof will depend upon thecharacteristics of the subject. Such characteristics include thecondition, disease severity, general health, age, sex and body weight ofthe subject. One of ordinary skill in the art will be readily able todetermine appropriate dosages depending on these and other relatedfactors. In addition, both objective and subjective assays mayoptionally be employed to identify optimal dosage ranges.

A therapeutically effective amount is commonly administered in a dosingregimen that may comprise multiple unit doses. For any particulartherapeutic protein, a therapeutically effective amount (and/or anappropriate unit dose within an effective dosing regimen) may vary, forexample, depending on route of administration, on combination with otherpharmaceutical agents. Also, the specific therapeutically effectiveamount (and/or unit dose) for any particular patient may depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific pharmaceutical agentemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and/or rate of excretion or metabolism of thespecific fusion protein employed; the duration of the treatment; andlike factors as is well known in the medical arts.

In some embodiments, the therapeutically effective dose ranges fromabout 0.005 mg/kg brain weight to 500 mg/kg brain weight, e.g., fromabout 0.005 mg/kg brain weight to 400 mg/kg brain weight, from about0.005 mg/kg brain weight to 300 mg/kg brain weight, from about 0.005mg/kg brain weight to 200 mg/kg brain weight, from about 0.005 mg/kgbrain weight to 100 mg/kg brain weight, from about 0.005 mg/kg brainweight to 90 mg/kg brain weight, from about 0.005 mg/kg brain weight to80 mg/kg brain weight, from about 0.005 mg/kg brain weight to 70 mg/kgbrain weight, from about 0.005 mg/kg brain weight to 60 mg/kg brainweight, from about 0.005 mg/kg brain weight to 50 mg/kg brain weight,from about 0.005 mg/kg brain weight to 40 mg/kg brain weight, from about0.005 mg/kg brain weight to 30 mg/kg brain weight, from about 0.005mg/kg brain weight to 25 mg/kg brain weight, from about 0.005 mg/kgbrain weight to 20 mg/kg brain weight, from about 0.005 mg/kg brainweight to 15 mg/kg brain weight, from about 0.005 mg/kg brain weight to10 mg/kg brain weight.

In some embodiments, the therapeutically effective dose is greater thanabout 0.1 mg/kg brain weight, greater than about 0.5 mg/kg brain weight,greater than about 1.0 mg/kg brain weight, greater than about 3 mg/kgbrain weight, greater than about 5 mg/kg brain weight, greater thanabout 10 mg/kg brain weight, greater than about 15 mg/kg brain weight,greater than about 20 mg/kg brain weight, greater than about 30 mg/kgbrain weight, greater than about 40 mg/kg brain weight, greater thanabout 50 mg/kg brain weight, greater than about 60 mg/kg brain weight,greater than about 70 mg/kg brain weight, greater than about 80 mg/kgbrain weight, greater than about 90 mg/kg brain weight, greater thanabout 100 mg/kg brain weight, greater than about 150 mg/kg brain weight,greater than about 200 mg/kg brain weight, greater than about 250 mg/kgbrain weight, greater than about 300 mg/kg brain weight, greater thanabout 350 mg/kg brain weight, greater than about 400 mg/kg brain weight,greater than about 450 mg/kg brain weight, greater than about 500 mg/kgbrain weight.

In some embodiments, the therapeutically effective dose may also bedefined by mg/kg body weight. As one skilled in the art wouldappreciate, the brain weights and body weights can be correlated.Dekaban A S. “Changes in brain weights during the span of human life:relation of brain weights to body heights and body weights,” Ann Neurol1978; 4:345-56. Thus, in some embodiments, the dosages can be convertedas shown in Table 5.

TABLE 5 Correlation between Brain Weights, body weights and ages ofmales Age (year) Brain weight (kg) Body weight (kg) 3 (31-43 months)1.27 15.55 4-5 1.30 19.46

In some embodiments, the therapeutically effective dose may also bedefined by mg/15 cc of CSF. As one skilled in the art would appreciate,therapeutically effective doses based on brain weights and body weightscan be converted to mg/15 cc of CSF. For example, the volume of CSF inadult humans is approximately 150 mL (Johanson C E, et al. “Multiplicityof cerebrospinal fluid functions: New challenges in health and disease,”Cerebrospinal Fluid Res. 2008 May 14; 5:10). Therefore, single doseinjections of 0.1 mg to 50 mg protein to adults would be approximately0.01 mg/15 cc of CSF (0.1 mg) to 5.0 mg/15 cc of CSF (50 mg) doses inadults.

It is to be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the enzyme replacement therapy andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed invention.

Kits

The present invention further provides kits or other articles ofmanufacture which contains the formulation of the present invention andprovides instructions for its reconstitution (if lyophilized) and/oruse. Kits or other articles of manufacture may include a container, anIDDD, a catheter and any other articles, devices or equipment useful ininterthecal administration and associated surgery. Suitable containersinclude, for example, bottles, vials, syringes (e.g., pre-filledsyringes), ampules, cartridges, reservoirs, or lyo-jects. The containermay be formed from a variety of materials such as glass or plastic. Insome embodiments, a container is a pre-filled syringe. Suitablepre-filled syringes include, but are not limited to, borosilicate glasssyringes with baked silicone coating, borosilicate glass syringes withsprayed silicone, or plastic resin syringes without silicone.

Typically, the container may holds formulations and a label on, orassociated with, the container that may indicate directions forreconstitution and/or use. For example, the label may indicate that theformulation is reconstituted to protein concentrations as describedabove. The label may further indicate that the formulation is useful orintended for, for example, IT administration. In some embodiments, acontainer may contain a single dose of a stable formulation containing atherapeutic agent (e.g., a replacement enzyme). In various embodiments,a single dose of the stable formulation is present in a volume of lessthan about 15 ml, 10 ml, 5.0 ml, 4.0 ml, 3.5 ml, 3.0 ml, 2.5 ml, 2.0 ml,1.5 ml, 1.0 ml, or 0.5 ml. Alternatively, a container holding theformulation may be a multi-use vial, which allows for repeatadministrations (e.g., from 2-6 administrations) of the formulation.Kits or other articles of manufacture may further include a secondcontainer comprising a suitable diluent (e.g., BWFI, saline, bufferedsaline). Upon mixing of the diluent and the formulation, the finalprotein concentration in the reconstituted formulation will generally beat least 1 mg/ml (e.g., at least 5 mg/ml, at least 10 mg/ml, at least 25mg/ml, at least 50 mg/ml, at least 75 mg/ml, at least 100 mg/ml). Kitsor other articles of manufacture may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, IDDDs, catheters, syringes, andpackage inserts with instructions for use.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature citations are incorporated byreference.

EXAMPLES Example 1: HNS Formulation

The experiments in the present example were designed as part of thepre-formulation study to examine the stability of Heparan-N-Sulfatase(HNS) in various formulation conditions including pH, ionic strength,and buffer type intended for intrathecal delivery.

HNS is generally found to be a dimer in its native state (Bielicki etal., Journal of Biochemistry, 1998, 329, 145-150). The molecular weightof the HNS dimer is 115 kDa. HNS typically elutes as a dimer during sizeexclusion chromatography (SEC). When run on SDS-PAGE gels, HNS appearsas a dimer unless the sample is heated to 100° C. prior to loading onthe gel, in which case it appears as a monomer (62 kDa). The full lengthand mature sequences of HNS are shown below in Table 6 and Table 7,respectively. The mature HNS sequence contains 5 cysteine residues(underlined), which could allow for two internal disulfide bonds and onefree cysteine.

TABLE 6 Sequence of Full Length HNSMSCPVPACCA LLLVLGLCRA RPRNALLLLA DDGGFESGAYNNSAIATPHL DALARRSLLF RNAFTSVSS C  SPSRASLLTGLPQHQNGMYG LHQDVHHFNS FDKVRSLPLL LSQAGVRTGIIGKKHVGPET VYPFDFAYTE ENGSVLQVGR NITRIKLLVR KFLQTQDDRP FFLYVAFHDP HR CGHSQPQY GTF C EKFGNG ESGMGRIPDW TPQAYDPLDV LVPYFVPNTP AARADLAAQYTTVGRMDQGV GLVLQELRDA GVLNDTLVIF TSDNGIPFPSGRTNLYWPGT AEPLLVSSPE HPKRWGQVSE AYVSLLDLTPTILDWFSIPY PSYAIFGSKT IHLTGRSLLP ALEAEPLWATVFGSQSHHEV TMSYPMRSVQ HRHFRLVHNL NFKMPFPIDQDFYVSPTFQD LLNRTTAGQP TGWYKDLRHY YYRARWELYDRSRDPHETQN LATDPRFAQL LEMLRDQLAK WQWETHDPWV C APDGVLEEK LSPQ CQPLHN EL (SEQ ID NO: 2)

TABLE 7 Sequence of Mature HNS (rhHNS)RPRNALLLLA DDGGFESGAY NNSAIATPHL DALARRSLLF RNAFTSVSS C  SPSRASLLTG LPQHQNGMYG LHQDVHHFNSFDKVRSLPLL LSQAGVRTGI IGKKHVGPET VYPFDFAYTEENGSVLQVGR NITRIKLLVR KFLQTQDDRP FFLYVAFHDP HR C GHSQPQY GTF CEKFGNG ESGMGRIPDW TPQAYDPLDV LVPYFVPNTP AARADLAAQY TTVGRMDQGV GLVLQELRDAGVLNDTLVIF TSDNGIPFPS GRTNLYWPGT AEPLLVSSPEHPKRWGQVSE AYVSLLDLTP TILDWFSIPY PSYAIFGSKTIHLTGRSLLP ALEAEPLWAT VFGSQSHHEV TMSYPMRSVQHRHFRLVHNL NFKMPFPIDQ DFYVSPTFQD LLNRTTAGQPTGWYKDLRHY YYRARWELYD RSRDPHETQN LATDPRFAQL LEMLRDQLAK WQWETHDPWV  CAPDGVLEEK LSPQ C QPLHN EL (SEQ ID NO: 1)

In this example, the following formulation parameters were examined: (1)pH in citrate formulations of pH 3-8 and in phosphate formulations of pH5-8; (2) Buffers: sodium citrate buffer (pH 3.0-8.0) and sodiumphosphate buffer (pH 5.0-8.0), all at 20 mM concentration; and (3) Ionicstrength: NaCl (0-300 mM).

All pre-formulation studies described in this example were conducted atlow protein concentrations of 1-2 mg/mL.

In order to analyze formulation products and degradation productsgenerated under various stresses, SEC-HPLC, SDS-PAGE, DifferentialScanning Calorimetry (DSC), turbidity (OD 320) and enzymatic activityassays were used.

Generally, SDS-PAGE results showed fragmentation of the formulation atlow pH (pH 3), while higher pH formulations showed little fragmentation.Evaluation of melting temperature by DSC showed rhHNS formulationscontaining citrate and phosphate have greatest thermal stability at a pHrange of 6-7. Enzymatic activity results showed that rhHNS formulationscontaining citrate at all pH values evaluated became inactive afterstorage at 50° C. for 7 days. rhHNS formulations containing phosphate atpH 6-7 retained significant activity after storage at 50° C. for 7 days.However, a high molecular weight peak (“16 minute peak”, as seen by SEC)is maximal at pH 7-8, although this peak was not consistently observedin separate preparations of the same formulation.

The effects of ionic strength, 0-300 mM NaCl, on rhHNS formulationstability were also evaluated. SDS-PAGE gels of samples stored ataccelerated stability conditions of 50° C. for 7 days showed no greaterfragmentation than of the internal lot control. rhHNS formulationscontaining citrate showed a complete loss in activity after 7 days at50° C. regardless of the ionic strength. rhHNS formulations containingphosphate retained significant activity in 50-300 mM NaCl. However, the16 minute peak (by SEC) is maximal in the 50-150 mM NaCl range.

Methods

Effect of pH on rhHNS Stability

rhHNS (9.2 mg/ml in 10 mM sodium phosphate, 138 mM sodium chloride, pH7.0) was buffer-exchanged using dialysis (Piece Slide-A-Lyzer, PN#66383,lot # HK107537) into 20 mM sodium citrate with a pH range of 3.0 to 8.0,and 20 mM sodium phosphate with a pH range of 6.0 to 8.0. The finalprotein concentration in each exchanged buffer was targeted to 2.0mg/mL. These solutions were aliquoted at 0.5 mL each into 2.0 ml glassvials (West Pharmeuticals, Cat#: 6800-0314, lot#: 30809A2001), and thenincubated in 50° C., 25° C. and 2-8° C. chambers. After 7, 14 and 28days, samples were pulled for analysis of aggregation (SEC-HPLC),fragmentation (SDS-PAGE), turbidity (OD320) and enzymatic activity.

The subsequent pH study in phosphate buffer was repeated following thesame procedures as above, however, rhHNS lot # SS10 was used. The pHrange for the initial phosphate study was narrow, so the study wasrepeated to incorporate a wider pH range.

OD320

Turbidity of rhHNS samples was determined by performing OD320measurements. Samples were measured in the Molecular Devices SpectraMaxPlus 384 at 2 mg/ml in a 0.2 cm pathlength cuvette. Total volume usedwas 30 μl for each testing.

SEC-HPLC

For SEC-HPLC analysis of rhHNS, a Superdex column 200 (10/300 GL, PN:17-5175-01, GE Healthcare) was used. The mobile phase was phosphatebuffered saline (25 mM sodium phosphate, 150 mM sodium chloride, pH 6)running at a flow rate of 0.5 ml/min. The injection volume was 30 μl of1 mg/ml (diluted from 2 mg/ml in respective buffer). The run time ofeach injection was 50 minutes and a detection wavelength of 214 nm.

SDS-PAGE

This method evaluates fragmentation and aggregation of rhHNS underreduced and denaturing conditions. rhHNS samples were mixed with SDSbuffer (final concentration=0.5 mg/ml), and DTT was added (reducedsamples only). Samples were heated to 100° C. for 5 minutes. Boilingsamples for longer than 5 minutes resulted in fragmentation of rhHNS.Each lane was loaded with 10 μg of rhHNS samples on 8-16% gradientacrylamide gels (Cat#: EC6045BOX). The gel was run at 150V and thenincubated overnight (with shaking) with Gel Code Blue Coomassie stain.The gels were destained with water for 1 hour prior to scanning.

Activity Assay

The activity assay for rhHNS is a two step reaction. In the firstreaction, heparan-N-sulfatase desulfates the substrate. Furtherhydrolysis occurs in the second reaction with the addition ofalpha-glucosidase enzyme that releases 4-MU, which can then be measured.rhHNS was diluted 1:210 for a final assay concentration of 10 μg/ml. Forthe Phosphate Buffer pH Study, the assay was modified and rhHNS wasdiluted 1:24 for a final assay concentration of ˜100 μg/mL.

DSC

Differential scanning calorimetry (DSC) measurements were made on theMicrocalorimeter instrument (MicroCal VP-DSC). rhHNS samples tested were0.5 mg/ml. The temperature was equilibrated to 10° C., and then rampedto 100° C. at 1° per minute.

Ionic Strength Effect on HNS Stability

rhHNS was buffer-exchanged using dialysis (Piece Slide-A-Lyzer lot #HK107537) into 20 mM citrate buffer of pH 6.0 with sodium chloride inthe range of 0-300 mM, and 20 mM phosphate buffer of pH 7.0 with sodiumchloride in the range of 0-300 mM. The final protein concentration ineach exchanged buffer was targeted to 2.0 mg/mL. These solutions werealiquoted at 0.5 mL each into 2.0 ml glass vials (West Pharmaceuticals,Cat#6800-0314, lot#30809A2001), and then incubated in 50° C., 25° C. and2-8° C. chambers. After 7, 14 and 28 days, samples were pulled foranalysis of aggregation (SEC-HPLC), fragmentation (SDS-PAGE), turbidity(OD 320), and enzymatic activity.

Results pH Effect on HNS Stability

OD320 and Appearance

The results of the OD 320 values to measure turbidity are shown below inTable 8. There were no significant changes in turbidity underaccelerated stability conditions of the rhHNS formulations containingphosphate at pH 7 or citrate at pH 3-6. However, the samples at pH 8.0in both phosphate and citrate formulations, and the citrate at pH 7.0showed increased turbidity after 7 days at 50° C. Appearance check wasperformed under a light box (M.W. Technologies, INC, Model #: MIH-DX)and all formulations appeared to remain clear, colorless and free ofvisible particulates.

TABLE 8 OD320 Summary of pH Study Storage Condition Formulation OD 320ILC 2 mg/ml HNS 20 mM Citrate, pH 3.0 0.005 50° C. 7 day 2 mg/ml HNS 20mM Citrate, pH 3.0 0.006 25° C. 14 day 2 mg/ml HNS 20 mM Citrate, pH 3.00.007  5° C. 28 day 2 mg/ml HNS 20 mM Citrate, pH 3.0 0.003 ILC 2 mg/mlHNS 20 mM Citrate, pH 4.0 0.002 50° C. 7 day 2 mg/ml HNS 20 mM Citrate,pH 4.0 0.006 25° C. 14 day 2 mg/ml HNS 20 mM Citrate, pH 4.0 0.009  5°C. 28 day 2 mg/ml HNS 20 mM Citrate, pH 4.0 0.000 ILC 2 mg/ml HNS 20 mMCitrate, pH 5.0 0.002 50° C. 7 day 2 mg/ml HNS 20 mM Citrate, pH 5.00.004 25° C. 14 day 2 mg/ml HNS 20 mM Citrate, pH 5.0 0.004  5° C. 28day 2 mg/ml HNS 20 mM Citrate, pH 5.0 0.004 ILC 2 mg/ml HNS 20 mMCitrate, pH 6.0 0.000 50° C. 7 day 2 mg/ml HNS 20 mM Citrate, pH 6.00.002 25° C. 14 day 2 mg/ml HNS 20 mM Citrate, pH 6.0 0.001  5° C. 28day 2 mg/ml HNS 20 mM Citrate, pH 6.0 0.000 ILC 2 mg/ml HNS 20 mMCitrate, pH 7.0 0.001 50° C. 7 day 2 mg/ml HNS 20 mM Citrate, pH 7.00.017 25° C. 14 day 2 mg/ml HNS 20 mM Citrate, pH 7.0 0.002  5° C. 28day 2 mg/ml HNS 20 mM Citrate, pH 7.0 −0.001 ILC 2 mg/ml HNS 20 mMCitrate, pH 8.0 0.000 50° C. 7 day 2 mg/ml HNS 20 mM Citrate, pH 8.00.018 25° C. 14 day 2 mg/ml HNS 20 mM Citrate, pH 8.0 0.002  5° C. 28day 2 mg/ml HNS 20 mM Citrate, pH 8.0 −0.001 ILC 2 mg/ml HNS 20 mMPhosphate, pH 7.0 0.000 50° C. 7 day 2 mg/ml HNS 20 mM Phosphate, pH 7.00.005 25° C. 14 day 2 mg/ml HNS 20 mM Phosphate, pH 7.0 0.000  5° C. 28day 2 mg/ml HNS 20 mM Phosphate, pH 7.0 0.001 ILC 2 mg/ml HNS 20 mMPhosphate, pH 8.0 0.002 50° C. 7 day 2 mg/ml HNS 20 mM Phosphate, pH 8.00.023 25° C. 14 day 2 mg/ml HNS 20 mM Phosphate, pH 8.0 0.002  5° C. 28day 2 mg/ml HNS 20 mM Phosphate, pH 8.0 0.001

SEC-HPLC

Representative chromatograms of SEC elution profiles of rhHNS are shownin FIGS. 1A-1C. The baseline sample mainly contains three peaks withretention times of ˜22 min, ˜26 min, and ˜32 min, respectively.Occasionally, it also has a peak at ˜34 min. The main peak at ˜26 min,was confirmed as a dimer by SEC-LS. The natures of other peaks areunknown.

The SEC data from the first pH study are summarized below in Table 9.Overall, all the formulations essentially had little change under thestressed conditions (50° C.) as well as accelerated (25° C.) and realtime storage condition (2-8° C.). However, after 7 days at 50° C., rhHNSformulations containing citrate or phosphate at pH 6-8 generated a highmolecular weight peak with a retention time of 16 min. In the rhHNSformulation containing phosphate pH 7.0, the 16 min peak accounts for˜2% of the total area. However, the same formulation prepared in theionic strength study only contained about 0.1%.

TABLE 9 SEC-HPLC Data Summary from pH Study Storage ConditionFormulation Description % 16 Min Peak % Dimer ILC 20 mM Citrate pH 3.0 099.8  7 day 50° C. 20 mM Citrate pH 3.0 0 99.9 14 day 25° C. 20 mMCitrate pH 3.0 0 99.7  1 mo 5° C. 20 mM Citrate pH 3.0 0 99.7 ILC 20 mMCitrate pH 4.0 0 99.8  7 day 50° C. 20 mM Citrate pH 4.0 0 99.8 14 day25° C. 20 mM Citrate pH 4.0 0 99.6  1 mo 5° C. 20 mM Citrate pH 4.0 099.5 ILC 20 mM Citrate pH 5.0 0 99.8  7 day 50° C. 20 mM Citrate pH 5.00 99.8 14 day 25° C. 20 mM Citrate pH 5.0 0 99.6  1 mo 5° C. 20 mMCitrate pH 5.0 0 99.5 ILC 20 mM Citrate pH 6.0 0 99.8  7 day 50° C. 20mM Citrate pH 6.0 0.3 99.0 14 day 25° C. 20 mM Citrate pH 6.0 0 99.6  1mo 5° C. 20 mM Citrate pH 6.0 0 99.3 ILC 20 mM Citrate pH 7.0 0 99.8  7day 50° C. 20 mM Citrate pH 7.0 0.9 98.6 14 day 25° C. 20 mM Citrate pH7.0 0 99.6  1 mo 5° C. 20 mM Citrate pH 7.0 0 99.2 ILC 20 mM Citrate pH8.0 0 99.8  7 day 50° C. 20 mM Citrate pH 8.0 0.5 98.7 14 day 25° C. 20mM Citrate pH 8.0 0 99.6  1 mo 5° C. 20 mM Citrate pH 8.0 0 99.4 ILC 20mM phosphate pH 7.0 0 99.8  7 day 50° C. 20 mM phosphate pH 7.0 2.0 97.214 day 25° C. 20 mM phosphate pH 7.0 0 99.6  1 mo 5° C. 20 mM phosphatepH 7.0 0 99.5 ILC 20 mM phosphate pH 8.0 0 99.8  7 day 50° C. 20 mMphosphate pH 8.0 1.0 98.8 14 day 25° C. 20 mM phosphate pH 8.0 0 99.7  1mo 5° C. 20 mM phosphate pH 8.0 0 99.4

In order to verify this phenomenon, the pH study in phosphate buffer wasrepeated over a wider range of pHs and the SEC data are summarized belowin Table 10. In this study, the 16 minute peak was not present in the pH5 buffer after 7 days at 50° C., but indeed existed in the formulationsof pH 6-8, which increases with increasing pH. Addition of polysorbate20 (0.05%) did not significantly affect the size of the 16 minute peak.Interestingly, although the pH 5 formulation did not contain this peakin the stability samples, during the preparation of dialyzing fromsaline solution into pH 5, a significant amount of rhHNS precipitated.

TABLE 10 SEC-HPLC Data Summary from Repeat Phosphate Buffer pH StudyStorage % 16 % Condition Formulation Description mm peak Dimer  5° C. 7days 2 mg/ml HNS 20 mM Phosphate, pH 5.0 0.0 98.4 50° C. 7 days 2 mg/mlHNS 20 mM Phosphate, pH 5.0 0.0 99.3  5° C. 7 days 2 mg/ml HNS 20 mMPhosphate, pH 6.0 0.0 99.0 50° C. 7 days 2 mg/ml HNS 20 mM Phosphate, pH6.0 0.2 99.1  5° C. 7 days 2 mg/ml HNS 20 mM Phosphate, pH 7.0 0.0 98.850° C. 7 days 2 mg/ml HNS 20 mM Phosphate, pH 7.0 0.6 98.8  5° C. 7 days2 mg/ml HNS 20 mM Phosphate, pH 7.0, 0.0 98.9 0.05% polysorbate 20 50°C. 7 days 2 mg/ml HNS 20 mM Phosphate, pH 7.0, 0.8 98.0 0.05%polysorbate 20  5° C. 7 days 2 mg/ml HNS 20 mM Phosphate, pH 8.0 0.099.1 50° C. 7 days 2 mg/ml HNS 20 mM Phosphate, pH 8.0 1.6 97.6

Preliminary characterization results confirm that the 16 minute peak hasa spectrum indicative of protein which, when scaled, superimposes wellwith the spectrum of rhHNS dimer peak (FIG. 1D). When examined bySEC-LS, the 16 minute peak displays an apparent molecular weight of >1MDa. Further characterization may be needed to understand the nature ofthis peak.

Enzyme Activity

The activity data summary from the first pH study is shown below inTable 5. Under accelerated stability conditions of 50° C. for 7 days,rhHNS lost most of the enzymatic activity in all citrate containingformulations, pH 3-8, while the rhHNS formulations containing phosphate,pH 7-8, retain activity. At 25° C. and 5° C., all rhHNS formulations inboth citrate and phosphate buffers retained most of the activity. rhHNSformulations containing citrate pH 3.0 appear to have lower overallactivity values. The activity data from the repeat pH study in phosphateare summarized in Tables 11 and 12. All rhHNS formulations in pH 5-7retain 84-100% of enzyme activity after 7 days at 50° C., except thatthe rhHNS formulation at pH 8.0 lost 65% activity.

TABLE 11 Activity Summary from the First pH Study Storage ConditionFormulation Description Activity nmol/mg/hr ILC 20 mM Citrate pH 3.0 639 7 day 50° C. 20 mM Citrate pH 3.0 29 14 day 25° C. 20 mM Citrate pH 3.0920  1 mo. 5° C. 20 mM Citrate pH 3.0 794 ILC 20 mM Citrate pH 4.0 2178 7 day 50° C. 20 mM Citrate pH 4.0 43 14 day 25° C. 20 mM Citrate pH 4.01998  1 mo. 5° C. 20 mM Citrate pH 4.0 2123 ILC 20 mM Citrate pH 5.01901  7 day 50° C. 20 mM Citrate pH 5.0 72 14 day 25° C. 20 mM CitratepH 5.0 1779  1 mo. 5° C. 20 mM Citrate pH 5.0 2194 ILC 20 mM Citrate pH6.0 2316  7 day 50° C. 20 mM Citrate pH 6.0 80 14 day 25° C. 20 mMCitrate pH 6.0 2026  1 mo. 5° C. 20 mM Citrate pH 6.0 2122 ILC 20 mMCitrate pH 7.0 2312  7 day 50° C. 20 mM Citrate pH 7.0 115 14 day 25° C.20 mM Citrate pH 7.0 2009  1 mo. 5° C. 20 mM Citrate pH 7.0 2205 ILC 20mM Citrate pH 8.0 2221  7 day 50° C. 20 mM Citrate pH 8.0 44 14 day 25°C. 20 mM Citrate pH 8.0 2071  1 mo. 5° C. 20 mM Citrate pH 8.0 2505 ILC20 mM phosphate pH 7.0 640  7 day 50° C. 20 mM phosphate pH 7.0 1200 14day 25° C. 20 mM phosphate pH 7.0 1749  1 mo. 5° C. 20 mM phosphate pH7.0 1391 ILC 20 mM phosphate pH 8.0 1451  7 day 50° C. 20 mM phosphatepH 8.0 1125 14 day 25° C. 20 mM phosphate pH 8.0 1620  1 mo. 5° C. 20 mMphosphate pH 8.0 1492

TABLE 12 Activity Summary from the Repeat pH Study Storage ActivityCondition Formulation Description nmol/mg/hr  ° C. 7 days 2 mg/ml HNS 20mM Phosphate, pH 5.0 4201 50° C. 7 days 2 mg/ml HNS 20 mM Phosphate, pH5.0 3952  5° C. 7 days 2 mg/ml HNS 20 mM Phosphate, pH 6.0 3923 50° C. 7days 2 mg/ml HNS 20 mM Phosphate, pH 6.0 4131  5° C. 7 days 2 mg/ml HNS20 mM Phosphate, pH 7.0 4107 50° C. 7 days 2 mg/ml HNS 20 mM Phosphate,pH 7.0 3841  5° C. 7 days 2 mg/ml HNS 20 mM Phosphate, pH 7.0, 49520.05% polysorbate 20 50° C. 7 days 2 mg/ml HNS 20 mM Phosphate, pH 7.0,4173 0.05% polysorbate 20  5° C. 7 days 2 mg/ml HNS 20 mM Phosphate, pH8.0 4729 50° C. 7 days 2 mg/ml HNS 20 mM Phosphate, pH 8.0 1590

SDS-PAGE Gels of HNS Formulations from pH Study

Exemplary SDS-PAGE gels are shown in FIG. 2 and FIG. 3. Reduced gelsfrom the pH study are shown in FIG. 2, and show fragmentation bands inthe formulation containing citrate at pH 3. All other formulations of pH4-8 were similar and show only a major (monomer) band at ˜60 kDa, exceptsome high MW aggregates shown in the pH 8.0 citrate buffer after 7 daysat 40° C. FIG. 3 shows the non-reduced gels from the pH study. Again,the fragmentation could be seen with the rhHNS formulation containingcitrate pH 3.

Based upon these results, it was apparent that the rhHNS native dimer isprimarily non-covalently bound since the presence or absence of areducing agent doesn't affect the position of the main band. A faint 125kDa dimer band, however, was present primarily in the non-reducedsamples suggesting that this band is a covalently bound non-nativedimer. The non-native dimer band was more pronounced in the ILC and 25°C. samples than in the 40° C. samples.

DSC Data from pH Study

FIG. 4 shows the pH dependant thermal stability of citrate as determinedby DSC. The highest melting temperature of rhHNS in citrate was 90° C.at pH 6.0. rhHNS formulations containing phosphate showed greatestthermal stability at pH 6-7. The melting temperature of rhHNS at everypH examined exceeded 70° C.

Ionic Strength Effect on rhHNS Stability

Turbidity and Appearance

The summary of OD320 values are shown below in Table 13. There was noobserved change in turbidity of the samples over time, and notemperature dependant change in values. The appearance of the samplesremained unchanged at each time point. All samples appeared clear,colorless, and no visible particulates

TABLE 13 OD320 of Ionic Effect Sample Formulation OD 320 ILC 10 mMCitrate pH 6.0 0.001 50° C. 7 day 10 mM Citrate pH 6.0 0.006 25° C. 14day 10 mM Citrate pH 6.0 0.005  5° C. 28 day 10 mM Citrate pH 6.0 0.003ILC 10 mM Citrate pH 6.0 100 mM NaCl 0.002 50° C. 7 day 10 mM Citrate pH6.0 100 mM NaCl 0.009 25° C. 14 day 10 mM Citrate pH 6.0 100 mM NaCl0.004  5° C. 28 day 10 mM Citrate pH 6.0 100 mM NaCl 0.001 ILC 10 mMCitrate pH 6.0 150 mM NaCl 0.001 50° C. 7 day 10 mM Citrate pH 6.0 150mM NaCl 0.007 25° C. 14 day 10 mM Citrate pH 6.0 150 mM NaCl 0.003  5°C. 28 day 10 mM Citrate pH 6.0 150 mM NaCl 0.001 ILC 10 mM Citrate pH6.0 300 mM NaCl 0.001 50° C. 7 day 10 mM Citrate pH 6.0 300 mM NaCl0.006 25° C. 14 day 10 mM Citrate pH 6.0 300 mM NaCl 0.006  5° C. 28 day10 mM Citrate pH 6.0 300 mM NaCl 0.003 ILC 10 mM Phosphate pH 7.0 0.00250° C. 7 day 10 mM Phosphate pH 7.0 0.009 25° C. 14 day 10 mM PhosphatepH 7.0 0.005  5° C. 28 day 10 mM Phosphate pH 7.0 0.002 ILC 10 mMPhosphate pH 7.0 50 mM NaCl 0.001 50° C. 7 day 10 mM Phosphate pH 7.0 50mM NaCl 0.009 25° C. 14 day 10 mM Phosphate pH 7.0 50 mM NaCl 0.003  5°C. 28 day 10 mM Phosphate pH 7.0 50 mM NaCl 0.002 ILC 10 mM Phosphate pH7.0 100 mM NaCl 0.002 50° C. 7 day 10 mM Phosphate pH 7.0 100 mM NaCl0.009 25° C. 14 day 10 mM Phosphate pH 7.0 100 mM NaCl 0.004  5° C. 28day 10 mM Phosphate pH 7.0 100 mM NaCl 0.002 ILC 10 mM Phosphate pH 7.0150 mM NaCl 0.002 50° C. 7 day 10 mM Phosphate pH 7.0 150 mM NaCl 0.00425° C. 14 day 10 mM Phosphate pH 7.0 150 mM NaCl 0.002  5° C. 28 day 10mM Phosphate pH 7.0 150 mM NaCl 0.001 ILC 10 mM Phosphate pH 7.0 300 mMNaCl 0.002 50° C. 7 day 10 mM Phosphate pH 7.0 300 mM NaCl 0.010 25° C.14 day 10 mM Phosphate pH 7.0 300 mM NaCl 0.005  5° C. 28 day 10 mMPhosphate pH 7.0 300 mM NaCl 0.003

SEC-HPLC

Table 14 shows the SEC-HPLC data summary from the ionic effect study.After 7 days at 50° C., all the formulations had little changes exceptthe 16 min peak. In the citrate buffer, the 16 minute peak area percentwas between 0.1 and 0.3% with no particular increasing or decreasingtrend with NaCl level. In phosphate buffer, however, there was anincrease in the 16 minute peak percent to ˜0.5% for ionic strengths from50 to 150 mM. At lower and higher ionic strengths, the 16 minute peakwent down to ˜0.1%.

TABLE 14 SEC-HPLC Data Summary of Ionic Strength Effect StorageCondition Formulation Description % 16 Min Peak % Dimer ILC Citrate pH6.0 0 99.3  7 day 50° C. Citrate pH 6.0 0.24 99.2 14 day 25° C. CitratepH 6.0 0 99.4  1 mo. 5° C. Citrate pH 6.0 0 99.5 ILC Citrate pH 6.0 100mM NaCl 0 99.3  7 day 50° C. Citrate pH 6.0 100 mM NaCl 0.10 99.3 14 day25° C. Citrate pH 6.0 100 mM NaCl 0 99.1  1 mo. 5° C. Citrate pH 6.0 100mM NaCl 0 99.4 ILC Citrate pH 6.0 150 mM NaCl 0 99.8  7 day 50° C.Citrate pH 6.0 150 mM NaCl 0.22 99.5 14 day 25° C. Citrate pH 6.0 150 mMNaCl 0 99.2  1 mo. 5° C. Citrate pH 6.0 150 mM NaCl 0 99.5 ILC CitratepH 6.0 300 mM NaCl 0 99.5  7 day 50° C. Citrate pH 6.0 300 mM NaCl 0.0499.6 14 day 25° C. Citrate pH 6.0 300 mM NaCl 0 99.4  1 mo. 5° C.Citrate pH 6.0 300 mM NaCl 0 99.3 ILC Phosphate pH 7.0 0 99.3  7 day 50°C. Phosphate pH 7.0 0.11 99.1 14 day 25° C. Phosphate pH 7.0 0 99.2  1mo. 5° C. Phosphate pH 7.0 0 99.4 ILC Phosphate pH 7.0 50 mM NaCl 0 99.4 7 day 50° C. Phosphate pH 7.0 50 mM NaCl 0.57 98.9 14 day 25° C.Phosphate pH 7.0 50 mM NaCl 0 99.3  1 mo. 5° C. Phosphate pH 7.0 50 mMNaCl 0 99.5 ILC Phosphate pH 7.0 100 mM NaCl 0 99.3  7 day 50° C.Phosphate pH 7.0 100 mM NaCl 0.53 98.9 14 day 25° C. Phosphate pH 7.0100 mM NaCl 0 99.2  1 mo. 5° C. Phosphate pH 7.0 100 mM NaCl 0 99.3 ILCPhosphate pH 7.0 150 mM NaCl 0 99.4  7 day 50° C. Phosphate pH 7.0 150mM NaCl 0.52 98.9 14 day 25° C. Phosphate pH 7.0 150 mM NaCl 0 99.2  1mo. 5° C. Phosphate pH 7.0 150 mM NaCl 0 99.4 ILC Phosphate pH 7.0 300mM NaCl 0 99.4  7 day 50° C. Phosphate pH 7.0 300 mM NaCl 0.06 99.3 14day 25° C. Phosphate pH 7.0 300 mM NaCl 0 99.3  1 mo. 5° C. Phosphate pH7.0 300 mM NaCl 0 99.1

Enzymatic Activity

Table 15 shows the activity data summary of ionic effect on rhHNSstability under accelerated conditions of 7 days at 50° C. rhHNSformulations containing citrate retained only 8-30% activity, regardlessof ionic strength under accelerated conditions. rhHNS formulationscontaining phosphate with 0-300 mM NaCl all showed higher overallactivity retained under accelerated conditions, 45-70%.

TABLE 15 Activity Summary of Ionic Strength Effect Study StorageActivity Condition Formulation Description nmol/mg/hr ILC Citrate pH 6.01725 7 day 50° C. Citrate pH 6.0 144 ILC Citrate pH 6.0 100 mM NaCl 17157 day 50° C. Citrate pH 6.0 100 mM NaCl 303 ILC Citrate pH 6.0 150 mMNaCl 1475 7 day 50° C. Citrate pH 6.0 150 mM NaCl 250 ILC Citrate pH 6.0300 mM NaCl 2059 7 day 50° C. Citrate pH 6.0 300 mM NaCl 619 ILCPhosphate pH 7.0 1661 7 day 50° C. Phosphate pH 7.0 766 ILC Phosphate pH7.0 50 mM NaCl 1441 7 day 50° C. Phosphate pH 7.0 50 mM NaCl 784 ILCPhosphate pH 7.0 100 mM NaCl 1290 7 day 50° C. Phosphate pH 7.0 100 mMNaCl 875 ILC Phosphate pH 7.0 150 mM NaCl 1297 7 day 50° C. Phosphate pH7.0 150 mM NaCl 839 ILC Phosphate pH 7.0 300 mM NaCl 1338 7 day 50° C.Phosphate pH 7.0 300 mM NaCl 693

SDS-PAGE

FIG. 6 shows silver stained SDS-PAGE gels of rhHNS formulations from theionic effects study after 7 days at 50° C. These gels were run using thesamples which were boiled for 10 minutes, so the fragmentation seen onthe gels could be due to 10 min boiling, since in the subsequentstudies, fragmentation was not observed when rhHNS was boiled for 5minutes.

The citrate formulations with 0-300 mM NaCl and phosphate formulationswith 0-300 mM NaCl all showed a primary monomer band at 60 kDa. Theinternal lot controls (stored at −80° C.) of rhHNS formulations appearedto show more pronounced fragment banding than samples held at 50° C. for7 days. Overall, the ionic strength did not affect banding pattern onSDS gels.

Conclusions

The results from the studies demonstrate that the primary stabilityindicating assays for the formulation screening are enzymatic activityand HPLC-SEC. DSC data showed that rhHNS has the greatest thermalstability with a Tm value of ˜90° C. at pH 6-7. rhHNS in the citratebuffers showed a significant loss of activity under acceleratedconditions at all pH's and ionic strengths, suggesting that citrate isan unacceptable formulation buffer. Results for phosphate wereconsiderably better, retaining maximal activity under acceleratedconditions at pH 6-7. Furthermore, phosphate formulations containing100-150 mM NaCl showed the greatest retention of activity underaccelerated conditions. The high molecular weight (16 minute) peak inSEC-HPLC, however, is maximal at pH 6-8 and at ionic strengths of 50-150mM, under accelerated conditions.

Additional formulation experiments are ongoing to better understand thecauses of the 16 minute peak. Furthermore, studies are ongoing tocompare the stability of phosphate formulations with un-buffer salineformulations, and the stability of low versus high proteinconcentration.

Example 2: Liquid Formulation for rhHNS

The experiments in this example were designed to optimize solubility ofrhHNS formulations intended for intrathecal delivery. As describedherein, intrathecal drug delivery requires a small amount of injectedliquid volume, and consequently, a highly concentrated protein solutionis needed. However, rhHNS typically has a heterogeneous charge profilewith an isoelectric point range from 5.1 to 6.5, which impacts itssolubility. The studies in the present example provide information onthe effect of pH and sodium chloride concentration on solubility of therhHNS product.

As can be seen in FIG. 7, increasing pH or salt concentration (e.g.,sodium chloride) resulted in increased rhHNS solubility. rhHNS nativestate was analyzed by analytical ultracentrifugation (AUC) of rhHNSformulated with varying salt concentration (145 mM or 300 mM). As can beseen in FIG. 8, rhHNS contains homogenous molecules and maintains thesame structure from 145 mM to 300 mM salt concentration at pH 7. Takentogether, these results indicated that increasing NaCl concentrationleads to increased solubility of rhHNS.

Two liquid formulations were identified for further study, including ahigh salt liquid formulation (15 mg/mL rhHNS, 175 mM NaCl, 5 mMphosphate, 0.005% polysorbate 20, pH 7.0); and a sucrose-containingformulation (15 mg/mL rhHNS, 2% sucrose, 145 mM NaCl, 5 mM phosphate,0.005% polysorbate 20, pH 7.0).

Example 3: Lyophilized Formulation for rhHNS

The experiments in this example were designed to optimize lyophilizationformulations and conditions for rhHNS. In particular, these studiesprovide information on the effect of formulation on the stability of theproduct, including appearance of the lyophilized cake, rhHNS enzymeactivity, and chemical integrity of the lyophilized product.

rhHNS was formulated into various phosphate based lyophilizedformulations. The following formulation parameters were examined: (1)Stabilizing Agent: Glucose (0.5-1%) or Sucrose (1-1.5%); and (2)Surfactant: Polysorbate 20 (0.02-0.005%). The following parameters wereused in all the formulations tested: (3) 15 mg/mL rhHNS; (4) 145 mMNaCl; (5) 5 mM phosphate; (6) pH 7.0.

Exemplary formulations were lyophilized according to the conditions inTable 16:

TABLE 16 Exemplary Lyophilization Cycle Total Lyo Time-4 daysFreezing/Annealing Freezing 0.25 C./min to −20 C. Hold −20 C. for 5 hrs.Continue Freezing −50 C. at 0.25 C./min Hold −50 for 3 hrs. PrimaryDrying Ramp 0.5 C./min to −25 C. and vacuum to 60 mT Hold −25 C. and 60mT for 50 hrs. Secondary Drying Ramp 0.3 C./min to 20 C. and N2 pressureto 150 mT Hold 20 C. and 150 mT for 6 hrs.

It was observed that glucose containing formulations had longreconstitution times (>30 minutes), although chemical stability wasmaintained (data not shown).

rhHNS lyo-formulations containing 1% sucrose had 15 month data at 2-8°C. and 3 month data at 25° C./40° C. (as shown below in Table 17),showing ≤1% change in SEC, RP, and SDS-PAGE.

TABLE 17 1% Sucrose Lyo-formulation Stability 5 ± 3° C. 25 ± 2° C. 40 ±2° C. Test Pre-Lyo Post-Lyo 3 M 15 M 3 M 0.5 M Cake Appearance NA White,White, White, White, White, solid cake solid cake solid cake solid cakesolid cake Moisture Content NA 0.5% NA NA NA NA Recon Time NA <60 sec<60 sec <60 sec <60 sec <60 sec Appearance of Opalescent, Opalescent,Opalescent, Opalescent, Opalescent, Opalescent, Recon solution colorlesscolorless colorless colorless colorless colorless w/o particles w/oparticles w/o particles w/o particles w/o particles w/o particles pH 7.27.2 7.3  NA 7.2 7.2 Protein Conc. 14.8 14.8 NA 15.3 14.6 14.0 (mg/mL)Specific Activity 71 62 NA 78 85 78 (U/mg) SEC main peak % 99.9% 99.8%99.6% 99.5% 99.6% 99.5% RP-HPLC Main 99.0% 98.9% 98.4% 98.5% 98.7% 98.5%peak % SDS-PAGE Conforms Conforms Conforms Slight Conforms Conformsincrease in lower band (<1%)

rhHNS lyo-formulations containing 1.5% sucrose had 14 month data at 2-8°C. and 3 month data at 25° C. (as shown below in Table 18), showing≤0.2% change in SEC, RP, and SDS-PAGE.

TABLE 18 1.5% Sucrose Lyo-formulation Stability 5 ± 3° C. 25 ± 2° C. 40± 2° C. Test Pre-Lyo Post-Lyo 3 M 3 M 1 M 3 M Cake Appearance NA White,White, White, White, White, solid cake solid cake solid cake solid cakesolid cake Moisture Content NA 0.5% NA NA NA NA Recon Time NA <60 sec<60 sec <60 sec <60 sec <60 sec Appearance of Opalescent, Opalescent,Opalescent, Opalescent, Opalescent, Opalescent, Recon solution colorlesscolorless colorless colorless colorless colorless w/o particles w/oparticles w/o particles w/o particles w/o particles w/o particles pH 6.96.9 6.9 6.9 6.9 6.9 Protein Conc. 16.1 14.2 15.7 15.4 16.6 15.4 (mg/mL)Specific Activity 176 210 149 122 169 139 (U/mg) SEC main peak % 99.9%99.8% 99.8% 99.6% 99.6% 99.0% RP-HPLC Main 99.2% 99.0% 98.8% 98.5% 98.0%97.7% peak % SDS-PAGE Conforms Conforms Conforms Slight Conforms Slightincrease in increase in lower band lower band (<1%) (~1%)

Lyophilized cakes were observed for cake appearance and integrity (e.g.,melt-back). As can be seen in FIG. 9A, lyophilized cakes formulated with1.5% sucrose had more cake shrinkage than those formulated with 1.0%sucrose. Lyophilized cakes formulated with 1.5% sucrose were also moresensitive to different lyophilization units, such as the VirTis unit vsthe LyoStar unit (FIG. 9B).

A separate set of experiments confirms that increasing sucrose causesincreased cake shrinkage, as indicated in Table 19 below.

TABLE 19 Comparison of rhHNS Stability and Cake Appearance at VariousSucrose Changes From the Baseline 1.25% Testing 1% Sucrose Sucrose 1.5%Sucrose Long Term  22M 12M 6M  21M  5M (5 C.) SEC   0.4%   0.2%   0.1%  0.1%     0% RP   0.5%   0.5%   0.5%   0.3%   0.5% SDS-PAGE   <1%   <1%  <1%     0%     0% Accelerated  20M 12M 6M  21M 12M (25 C.)   0.5%  0.2%   0.1%   0.1%     0% SEC   1.5%   0.5%   0.5%   0.5%     0% RP  <1%     1%     1%     0%     0% SDS-PAGE Stress Data 0.5M 1M 3M 1M0.5M  1M (40 C.)   0.5%   0.4%     1%   0.1%   0.1%   0.2% SEC   0.5%  0.5%   1.5%   0.1%   0.5%   0.2% RP   <1%   <1%   >1%     1%     0%    0% SDS-PAGE Cake Slight shrinkage Some More Appearance shrinkageshrinkage

Taken together, these data demonstrate that an increase in sucroseconcentration in rhHNS lyo-formulations correlated with an increase instability as well as an increase in lyophilized cake shrinkage.

Reconstituted lyo-formulations were observed for the presence ofparticulates by Micro-Flow Imaging (MFI). Exemplary particulate imagesare depicted in FIG. 10. As can be seen in FIG. 10, large particles wereobserved after reconsistution of lyo-formulations containing either 1%and 1.5% sucrose after storage.

Prelyophilized formulations were observed for the presence ofparticulates after 0.22 um filtration. As can be seen in FIG. 11, thepresence of polysorbate 20 (P20) prevents protein-like flocculants thatwere generated without P20 upon 0.22 μm filtration. Thus, P20 iseffective in preventing particulate formation and/or protecting rhHNSprotein during filtration. Further studies showed that the presence ofP20 was effective in reducing the presence of freeze-thaw inducedparticulates as well as lyophilization-induced particulates in rhHNSformulations (data not shown).

Lyophilization Conditions

Lyophilization cycle conditions were studied to determine the effect onrhHNS lyophilized formulations. For example, primary drying temperatureswere varied from −38 C to −20 C, and stability of rhHNS lyo-formulationswere determined by enzyme activity, SEC, RP, and cake appearance.Exemplary results of these analyses are shown in Table 20 below.

TABLE 20 Effect of Primary Drying on 1.5% Sucrose Lyo-formulations (−38°C.) (−30° C.) (−25° C.) (−23° C.) (−20° C.) 40° C. 40° C. 40° C. 40° C.40° C. Test Pre-Lyo Post-Lyo 0.5 M Post-Lyo 1 M Post-Lyo 0.5 M Post-Lyo0.5 M Post-Lyo 0.5 M Activity 246 285 236 227 125 141 124 160 128 249157 SEC 99.8 99.8 99.7 99.7 99.7 99.7 99.7 99.7 99.7 99.6 99.8 RP 98.898.8 98.5 98.8 NT 99.2 NT 99.1 98.8 98.9 99.0 2-10 μm NT 2958 1395 75934013 1550 3188 869 942 1235 4650 Cake NA — — — — — — — — — — Shrinkage

As can be seen, no significant difference in stability profile wasobserved within a range of primary drying temperature from −38 C to −20C. Lyophilized cake appearance showed increased cake shrinkage at aprimary drying temperature of −20 C. Similar results were also observedin lyo-formulations containing 1.25% and 1.0% sucrose (data not shown).

Example 4. Chronic Intrathecal Administration of Heparan N-Sulfatase

This example demonstrates that intrathecal administration can be used toeffectively deliver a lysosomal enzyme, such as recombinant humanheparan N-sulfatase (rhHNS), into brain tissues for the treatment of theneurologic symptoms of mucopolysaccharidosis IIIA (MPS IIIA; Sanfilipposyndrome), the defining clinical feature of this disorder. Experimentsdescribed in this example demonstrate that chronic IT administration ofrhHNS was well tolerated with dose-related enzyme activity detected inthe brain, spinal cord and liver.

In summary, an intrathecal (IT) formulation of recombinant human heparanN-sulfatase (rhHNS) has been developed for the treatment of theneurologic symptoms of mucopolysaccharidosis IIIA (MPS IIIA; Sanfilipposyndrome), the defining clinical feature of this disorder. Since theaverage age of MPS IIIA patients is 4.5 years, the pivotal toxicologystudies for rhHNS were conducted in juvenile cynomolgus monkeys toevaluate the effects on the developing brain. Monkeys were implantedwith an intrathecal (IT)-lumbar drug delivery device and dosed everyother week by short-term infusion (1.5, 4.5, or 8.3 mg/dose rhHNS for 6months; 12 doses), with device and vehicle controls receivingphosphate-buffered saline or vehicle, respectively. Eight animals pergroup (4/sex) were necropsied at 3 and 6 months (device-control groupnecropsied at 3 months), and 8 animals from the vehicle group and the 3rhHNS dose groups were necropsied 1 month after the final IT dose. NorhHNS-related clinical signs or gross central nervous system lesionswere observed. Compared to controls, there were cellular infiltrates ofslight-to-minimal mean severity in the meninges/perineurium surroundingthe brain/spinal cord correlating with transient increases incerebrospinal fluid (CSF) leukocytes, predominantly eosinophils, whichlargely resolved 1-month post-final dose. These changes were notassociated with any adverse morphologic changes in the brain or spinalcord. There appeared to be a dose related trend toward higher mean CSFrhHNS levels and in tissue rhHNS activity levels in the brain, spinalcord, and liver. The no-observed-adverse-effect-level was 8.3 mg/dosegiven every other week, the highest dose administered, indicating thatrhHNS may be safely administered intrathecally at various concentrationincluding concentrations higher than 8.3 mg/dose.

Sanfilippo A Disease

Mucopolysaccharidosis type IIIA (MPS IIIA; Sanfilippo A disease), a rarelysosomal storage disorder affecting approximately 1 in 100,000 peopleworldwide, results from the absence or defective function of heparanN-sulfatase (HNS) (Neufeld E F, et al. The Metabolic and Molecular Basesof Inherited Disease (2001) pp. 3421-3452), an exosulfatase involved inthe lysosomal catabolism of glycosaminoglycan (GAG) heparan sulfate. Inthe absence of this enzyme, GAG heparan sulfate accumulates in lysosomesof neurons and glial cells, with lesser accumulation outside the brain.The defining clinical feature of this disorder is central nervous system(CNS) degeneration, which results in loss of, or failure to attain,major developmental milestones. The progressive cognitive declineculminates in dementia and premature mortality.

IT Delivery of rhHNS

Since the average age of MPS IIIA patients is 4.5 years, the pivotaltoxicology studies for rhHNS were conducted in juvenile cynomolgusmonkeys (species selection based upon genetic and anatomic similarity tohumans) to evaluate the effects on the developing brain. The ageequivalence of monkeys to humans as cited in the literature ranges from7.6 months to 12.1 months for children 30 to 40 months old (Hood R D,Developmental and Reproductive Toxicology: A practical approach (2006)p. 276). As part of this effort, a 6-month toxicology study wasconducted in juvenile cynomolgus monkeys to evaluate IT lumbaradministration of rhHNS. The data obtained from a prior 1-month juvenilecynomolgus monkey toxicity study guided the dose level selection anddesign of the 6-month repeated-dose juvenile monkey study. Based upondata known to date, this is the first study involving the chronic ITadministration of ERT in juvenile nonhuman primates.

Fifty-six male and 56 female juvenile cynomolgus monkeys (Macacafascicularis) approximately 6 to 9 months old and weighing 0.82 to 1.81kg were used in this study. Monkeys were fed 15 biscuits ofPMI-Certified Primate Diet 5048 (Richmond, Ind.) daily. Water wasprovided ad libitum via a filtered automatic water system and waswithheld during urine collection periods. Monkeys were group-housed (twoper cage) for 2 to 4 weeks in stainless steel cages upon arrival withthe exception of the 3-month monkeys; these were individually housed instainless steel cages. For the duration of the study, all monkeys werehoused in individual stainless steel cages in rooms with controlledtemperature and humidity with a cycle of 12 hours of light and 12 hoursof darkness.

Prior to study initiation, all monkeys were implanted surgically with SCports and IT catheters. Prednisolone sodium succinate (IV, 30 mg/kg) andflunixin meglumine (intramuscular [IM], 2 mg/kg) were administered priorto surgery. The monkeys were pretreated with SC atropine sulfate (0.04mg/kg), sedated with IM ketamine HCl; 8 mg/kg), intubated, andmaintained on approximately 1 L/min of oxygen and 2.0% isoflurane. Anincision was made over the dorsal processes of the lumbar spine (L₄, L₅,or L₆), and a hemilaminectomy was made for the insertion of a taperedpolyurethane catheter (25 cm in length, 0.9 mm outer diameter×0.5 mminner diameter, with six side holes of 0.33 mm diameter) at L₃, L₄, orL₅. The catheter was inserted through a small dural incision and wasadvanced approximately 10 cm anterograde to the area of thethoracolumbar junction. A titanium SC port was attached to the ITcatheter and implanted in the SC tissue. Proper catheter placement wasconfirmed by myelogram using Isovue-300 (0.8 ml; Bracco Diagnostics,Inc., Princeton, N.J.). After recovering from surgery, monkeys receivedbutorphanol tartrate (IM, 0.05 mg/kg) and ceftiofur sodium (IM, 5.0mg/kg twice daily for 2 days).

In this example, rhHNS was provided in an IT formulation vehicleincluding 5 mM sodium phosphate, 145 mM sodium chloride, and 0.005%polysorbate 20 (pH 7.0). EOW doses of rhHNS were administered as ashort-term infusion over approximately eleven minutes: 0.6 mL (4minutes) followed with a flush of 0.5 mL phosphate-buffered saline (PBS)(7 minutes). Monkeys in the vehicle-control group received the ITformulation alone; DC monkeys received PBS (pH 7.2) IT.

Morbidity and Mortality

There were no rhHNS-related deaths or early sacrifices. There were norhHNS-related clinical signs noted at dosing or during the dailyobservations. Misplacement, pruritis, tremors, and ataxia observedduring and after dosing resolved within a few minutes to approximately 4hours of administration, and were considered a volume-related responserather than a reaction to rhHNS or the vehicle. Clinical signs observedduring and immediately after dosing were seen at a comparable incidencein control groups (DC and/or vehicle-dosed group); there was no evidenceof a dose response. In general, the incidence of clinical signs atdosing decreased with each subsequent dose. There were no rhHNS-relatedchanges in body weight, food consumption, and physical and neurologicfindings, or alterations in ECG or ophthalmology examinations.

Clinical Pathology

There were no changes considered related to rhHNS in hematology, serumchemistry, coagulation, or urinalysis parameters at any interval.

CSF Cell Counts and Chemistry

There were dose-related increases in mean CSF leukocyte counts for allgroups, including DC and 0 mg/dose groups, 24 hours postdose. There wasa general increase in leukocyte counts with each dose administered.Collection of CSF from approximately one half of the monkeys prior todosing showed that these effects had abated in the 2 weeks since theprevious dose. After dose 5, in addition to an increase in leukocytes,higher group mean CSF total protein and albumin were observed for therhHNS-dosed males in the 4.5 and 8.3 mg/dose groups (up to 4- to 5-fold)compared with the predose mean (P≤0.05 versus the DC and the 0 mg/dosegroup); less of a trend was evident in the female rhHNS-dosed groups.

rhHNS Concentrations and Antibody Analysis

Typically, the mean rhHNS levels in serum were <limit of detection (LOD)for all test groups for all time points. The rhHNS concentration in CSFfrom monkeys in the DC- and vehicle-dosed control group was generallybelow the limit of quantification (LOQ). Although no statisticalanalyses were performed, there appeared to be a dose-related trendtowards higher mean rhHNS levels in CSF in the 1.5, 4.5, and 8.3 mg/dosegroups. The predose CSF mean rhHNS levels were significantly lower thanthe postdose CSF levels. The mean rhHNS concentrations for the 6-monthcohort (both sexes) at study termination (main and recovery necropsy)are sumarized in Table 21. At a given dose level, mean concentrations ofrhHNS in the CSF appeared to be maintained in the same range (FIG. 12A)despite the anti-HNS antibody levels in the serum and CSF, whichcontinued to rise throughout the study.

TABLE 21 CSF rhHNS concentrations at study termination (main andrecovery necropsies). Main Necropsy Recovery Necropsy Mean ± SD^(a) Mean± SD Group n (ng/mL) n (ng/mL) Vehicle 8 — 8 NA 1.5 mg IT 8 516,366 ±1,024,084 8 NA 4.5 mg IT 7 377,460 ± 304,996 7 NA 8.3 mg IT 8 419,492 ±345,975 8 NA CSF, cerebrospinal fluid; HNS, human heparan N-sulfatase; n= number of samples above the LOQ; IT, intrathecal; SD, standarddeviation. ^(a) = samples collected approx. 24 hours postdose. NA = nosamples available for analysis or samples below the LOQ.

In the 6-month/recovery cohort, none of the monkeys in the devicecontrol group (PBS only) or those dosed with vehicle developed anti-HNSantibodies in serum or CSF at any time point tested. All monkeys in the1.5, 4.5, and 8.3 mg/dose groups tested negative (<LOD) for anti-HNSantibodies in serum and CSF samples collected prestudy (for CSF) and atpredose 2. By the end of the study, all monkeys tested positive foranti-HNS antibodies in serum.

All monkeys in the 1.5 mg/dose and 8.3 mg/dose groups and six of eightmonkeys in the 4.5 mg/dose group tested positive for anti-HNS antibodiesin the CSF at one or more time points. Since two monkeys in the 4.5 mggroup had no sample collected at any time point including necropsy,these results would appear to indicate that all monkeys dosed with rhHNSproduced an antibody response.

At all three dose levels, anti-HNS antibody concentrations in serum weredetected after dose 2, and levels increased markedly after dose 4.Although no statistical analyses were performed, there appeared to be adose-related trend towards higher serum antibody concentration; by theend of the study, levels were comparable across the 3 rhHNS dose groups(FIG. 12B). Anti-HNS antibody levels in the serum were always higherthan in the CSF over the time course of this study (from 9 to 236-foldserum/CSF antibody concentrations); the highest ratios of serum to CSFconcentrations (98 and 236-fold) were seen at 8.3 mg dose level in theearlier course of dosing (6 and 10 weeks).

Anti-HNS antibody concentrations in the serum increased 9-, 16-, and16-fold at 1.5 mg, 4.5 mg, and 8.3 mg/dose levels, respectively, in theearly time of dosing (from week 6 to week 14). During the same timeperiod, CSF antibody concentrations increased 30-, 41-, and 52-fold at1.5 mg, 4.5 mg, and 8.3 mg/dose levels, respectively (FIG. 12B);substantial levels remained after the 1-month dose-free recovery phase(Table 22).

TABLE 22 CSF anti-HNS antibody concentrations at study termination (mainand recovery necropsies). Main Necropsy^(a) Recovery Necropsy Mean ± SDMean ± SD Group n (ng/mL) n (ng/mL) Vehicle 8 — 8 — 1.5 mg IT 8 351,456± 244,171 8 299,512 ± 226,654 4.5 mg IT 7 147,187 ± 213,095 7 193,045 ±157,896 8.3 mg IT 8 185,227 ± 315,858 8 238,727 ± 185,785 CSF,cerebrospinal fluid; HNS, human heparan N-sulfatase; IT, intrathecal; n,number of sample above the limit of quantification; SD, standarddeviation. ^(a)Samples collected approximately 1 week prior to dosing.

Anti-HNS antibodies appeared later in the CSF than in serum (FIG. 12C).No apparent dose-related differences of antibody concentrations in theserum or CSF were observed (statistical analysis was not done due tosmall sample sizes); there was no observable difference between malesand females in antibody responses.

In the presence of anti-HNS antibody in the CSF, the mean concentrationsof rhHNS in the CSF appeared to be maintained, suggesting that thepresence of anti-HNS antibodies in the serum and CSF did not alter theconcentration level of the IT-dosed rhHNS. The 6-month/recovery cohortanalyses of the 6-month repeat-dose administration of rhHNS indicatedthat the anti-HNS antibody concentrations for the 3-month interim and6-month cohort sacrifice monkeys were comparable (FIG. 12C).

Gross and Histopathologic Findings

At all dose levels (although not at all sacrifice intervals,gender-specific, nor in a dose-related manner), eosinophilic infiltrateswere present in the parenchyma of the brain (predominantly gray matter),spinal cord (gray and white matter), dorsal spinal nerve roots/gangliaand the trigeminal ganglia (mid-dose males only) (FIGS. 13A-E). Theinfiltrates appeared to be secondary to the meningeal/perineuriuminfiltrates and/or to the presence of (penetration by) rhHNS within theparenchyma of the tissue. Although there were numerous inflammatory typechanges, the monkeys appeared to tolerate administration of rhHNS andnone of the infiltrates were considered related to or causing adversemorphologic changes in the nervous system parenchyma. Specifically,there was no evidence of neuronal necrosis/degeneration and no glialresponse related to rhHNS administration.

Microgliosis in the gray matter of the brain and spinal cord, inassociation with cellular infiltrates, predominantly eosinophilic, wasrelatively common in a previously performed 1-month juvenile monkeytoxicity study; these changes were relatively uncommon by the 3-monthinterim sacrifice in the 6-month study, but residual evidence of such aresponse could still seen in the 6-month cohort (FIG. 13F). Microglialreactions tend to be a relatively early event in the reaction to some(typically protein-based) centrally administered (or centrally-reactive)test articles. The eosinophilic infiltrates did correlate with increasednumber of eosinophils in the CSF of rhHNS-dosed monkeys, although thecells were not present in sufficient numbers to elicit an adversereaction.

At all dose levels, eosinophilic infiltrates were observed in the dorsalspinal nerve roots/ganglia for most rhHNS-dosed groups, regardless ofgender. The infiltrates in the various nervous system tissues appearedto be secondary to the meningeal/perineurium infiltrates and/or to thepresence of (penetration by) rhHNS within the parenchyma of the tissue.In the recovery sacrifice monkeys, rhHNS-related effects were generallyeither absent or reduced to control levels. Some changes, such asmicrogliosis in the spinal cord, were completely resolved after therecovery period. None of the rhHNS-related changes appeared to beassociated with any adverse structural microscopic changes in the brainor spinal cord. There was no neuronal necrosis noted in the brain,spinal cord, or ganglia.

Nerve fiber degeneration and gliosis in the spinal cord appeared to besecondary to the placement and/or presence of the IT catheter. Thesechanges were relatively similar between the control and rhHNS-dosedgroups. In the spinal nerve roots, Schwann cell (the myelinating cell ofthe peripheral nervous system) hyperplasia and nerve fiber degenerationwere present in both control and rhHNS-dosed monkeys. These changes weredue to damage to one or more spinal nerve roots at the time of catheterplacement.

HNS Enzyme Activity

In the 6-month/recovery cohorts, rhHNS enzyme activity in the spinalcord and brain of the vehicle-dosed group (0.0-0.154 nmol/hr/mg protein)were similar to levels shown in tissues from the 3-month interim cohort(0.0-0.0.154 nmol/hr/mg protein). Enzyme activity levels in the spinewere higher (approximately an order of magnitude higher in the lumbarspine) than levels measured in brain or liver, the 4.5 mg and 8.3mg/dose groups having similar levels. The rhHNS enzyme activity inspinal cord slices ranged from 3.9-18.6, 13.1-67.1, and 3.6-69.2nmol/hr/mg protein in males (FIG. 14A) and 1.8-16.2, 4.5-61.2, and21.1-66.0 nmol/hr/mg protein in females (FIG. 14B) for the 1.5, 4.5, and8.3 mg/dose groups, respectively. In spinal tissue after a 1-monthrecovery period, enzyme activity levels returned to levels consistentwith vehicle control values.

The rhHNS enzyme activity in brain slices ranged from 0.03-16.0,0.30-55.7, and 0.15-21.2 nmol/hr/mg protein in males (FIG. 14C), and0.04-5.1, 0.0-14.4 and 0.9-33.2 nmol/hr/mg protein in females (FIG. 14D)for the 1.5, 4.5, and 8.3 mg/dose groups, respectively. In brain tissueafter recovery, enzyme activity levels returned to levels consistentwith control values.

The fold-change in activity for different areas of the brain comparedwith endogenous levels (DC group) is shown in FIG. 15A. Although a trendtoward increased distribution was noted in surface samples, lumbar-ITadministered rhHNS could be shown to penetrate to periventricular areasof the brain.

In the 6-month cohort/recovery cohorts, mean activity levels in liverwere 0.50, 2.41, and 6.65 nmol/hr/mg protein in males and 1.04, 4.15,and 7.62 nmol/hr/mg protein in females for the 1.5, 4.5, and 8.3 mg/dosegroups, respectively (FIG. 15B). Levels in vehicle control monkeys were0.089 nmol/hr/mg protein for males and 0.083 nmol/hr/mg protein forfemales. Following the recovery period, rhHNS activity levels in liverwere comparable to baseline control levels for all dose groups.

Immunohistochemistry

rhHNS delivery to the CNS via bolus IT injection in the 3-month interimand 6-month/recovery cohorts resulted in delivery of immunoreactive testarticle to the pia-arachnoid tissues of the spinal cord and brain. Inthe monkeys that received IT rhHNS, the immunoreactive material wasconsistently present in meningeal and perivascular macrophages(brain/spinal cord) and variably present in the adjacent glial andneuronal cell populations. The lack of staining in vehicle-dosed controlmonkeys (FIG. 16A) demonstrated the specificity of the antibody to humanHNS. Generally, the immunoreactivity was dose related (i.e., using asemi-quantitative grading scale, increased immunohistochemical stainingwas noted in a generally dose-dependent manner). rhHNS delivery to theCNS via bolus IT resulted in positive immunostaining in the cerebralcortex and cerebellum (FIGS. 16B-D); however, immunoreactivity was notconsistently evident in the caudate/putamen region, midbrain, or deeperregions of the pons or medulla. Immunoreactivity was evident in thelivers (in sinusoidal lining cells including Kupffer cells, but not inhepatocytes) of all monkeys administered rhHNS. Immunoreactivity was notevident in the one female sacrificed early (4.5 mg/dose group) becauseof a leaking catheter that could not be repaired.

In the 1.5 mg/dose group, essentially full recovery was evident with theexception of liver and the meninges of the brain and spinal cord wheresome residual immunoreactivity was evident. At higher doses (4.5 and 8.3mg/dose), the intensity and incidences of immunoreactivity were lowerthan at the end of dosing. At all dose levels, the levels of rhHNS inspinal cord, brain, and liver approximated those seen in vehicle-dosedcontrols after the 1-month recovery period.

Discussion

In this study, EOW delivery of rhHNS administered IT for 6 months wasgenerally well tolerated. No remarkable changes were observed in bodyweight, clinical status, ophthalmologic/neurologic/physicalexaminations, ECGs, organ weights, or gross organ appearance. Findingswere limited to transient changes in CSF clinical pathology accompaniedby slight to mild meningeal infiltrates and epidural inflammation, withnearly complete reversal in all but the highest dose group following therecovery period. Widespread distribution of rhHNS throughout the brainand spinal cord was observed.

IT administration of rhHNS EOW elicited an inflammatory responsecharacterized by residual leukocyte infiltration and effusion of albuminnoted at 24 hours postdose and at necropsy. Without wishing to be boundby any particular theory, this presumably reflects a transient,localized, and incomplete opening of the BBB related to changes in thetight junctions near the catheter tip, resulting in entry of leukocytesand plasma proteins into the CSF (Simard J M, et al. Lancet Neurol.(2007) 6, 258-268; Stamatovic S M, et al. Curr. Neuropharmacol. (2008)6, 179-192). This may be the result of two components: one related tothe dose administration procedures or volume and another related to ITadministration of a protein.

The transient changes in BBB permeability (no significant differencesbetween dose groups and controls 24 hours postdose at the mainnecropsy), were not accompanied by any clinical signs.

There appeared to be a dose-related trend for higher mean CSF rhHNSlevels; at a given dose level, mean concentrations of rhHNS in the CSFappeared to be maintained in the same range despite the increasinganti-HNS antibody levels in the serum and CSF.

Meningeal cellular infiltration of slight-to-minimal mean severity wasobserved in the brains and spinal cords of rhHNS-dosed juvenile monkeys.This microscopic change was also noted in vehicle-dosed controls,indicating some of the response was related to IT catheter placement, aswell as a nonspecific inflammatory response to foreign protein. Theintroduction of a biologic/protein into the IT space, especially onethat penetrates the CNS, nearly always elicits some degree of aninflammatory response (Hovland D N, et al. Toxicol. Pathol. (2007) 35,1013-1029; Butt M T, Toxicol. Pathol. (2011) 39, 213-219), which, ifpresent in numbers that damage adjacent tissue, would represent anadverse effect. In the current study, however, these cells(predominantly eosionophils) appeared to represent a marker of tissuereaction/penetration and were not found in sufficient quantities toqualify as an adverse effect. None of the rhHNS-related changes appearedto be associated with any adverse structural microscopic changes in thebrain or spinal cord. There was no neuronal necrosis noted in the brain,spinal cord, or ganglia.

There were changes in the dorsal tracts of the spinal cord associatedwith the drug delivery device in some monkeys that included nerve fiberdegeneration, catheter tract fibrosis, and compression of the spinalcord; none of these changes were considered to be rhHNS-related in thatthey occurred in proximity to the IT catheter. The IT lumbar drugdelivery device was not specifically designed for IT implantation injuvenile monkeys, which have a smaller IT space than humans. Aretrospective analysis of microscopic evaluation data from control(device and/or saline-dosed) animals in IT studies concluded that someminimal degree of meningeal infiltration and catheter tract-associatedinflammation, fibrosis, and gliosis, and spinal cord nerve fiberdegeneration is seen (Butt M T, Toxicol. Pathol. (2011) 39, 213-219).

Evaluation of anti-test article antibodies is an important aspect of thetoxicity studies because of the potential impact of neutralizing orbinding antibodies on the clearance or biodistribution of test article(Ponce R P, et al. Regul. Toxicol. Pharmacol. (2009) 54, 164-182). Inthis study, since dose-related and quantitatively similar levels ofrhHNS enzyme activity were noted in the brain and spinal cord of the3-month interim and 6-month cohorts, and mean concentrations of rhHNS inthe CSF appeared to be maintained in the same range despite theincreasing anti-HNS antibody levels in the serum and CSF, we concludedthat no neutralizing activity was seen.

There appeared to be a dose-related trend toward higher levels of rhHNSenzyme activity in spinal cord, brain, and liver, that was highest nearthe injection site in the lumbar region of the spinal cord and uniformin the brain, with no significant differences rostral to caudal andbetween right and left hemispheres. No evidence for rhHNS accumulationwas noted in the brain and spinal cord tissue of the 6-month cohort ascompared with the 3-month interim cohort. Although a trend towardincreased distribution was noted in surface samples, lumbar-ITadministered rhHNS penetrated to deep, periventricular areas of thebrain. The rhHNS enzyme activity in the liver suggested the rhHNSredistributed systemically after IT delivery; no rhHNS-related adverseeffects were observed in the liver after evaluation of clinical andanatomic pathology parameters in the pivotal toxicity studies.

In general, the immunohistochemistry results corroborated the tissueenzyme activity in that dose-related immunoreactivity was observed inthe spinal cord and brain pia-arachnoid meninges and in the nervoustissues (neurons, glial cells) in the immediate proximity of themeninges. There was good gray matter penetration of the cerebrum andcerebellum after bolus IT injection or short-term IT infusion. Althoughimmunoreactivity was not evident in deeper structures such as the basalganglia or the central regions of the thalamus/hypothalamus, midbrain orthe pons/medulla, enzyme activity results indicate that lumbar-ITadministered rhHNS penetrated to deep, periventricular areas of thebrain. Thus, immunohistochemistry may be less sensitive technique fordetecting biodistribution of a test article. Immunoreactivity wasevident in Kupffer cells and the endothelial cells (cells capable ofphagocytosis) of the liver, but not parenchymal cells (hepatocytes).

The 6-month/recovery cohort analyses of the 6-month repeated-dose ITtoxicity study in juvenile monkeys indicated that rhHNS-related changesin the 3-month interim and 6-month sacrifice monkeys were comparable,including in-life parameters, clinical and anatomic pathology,concentrations of rhHNS and anti-HNS antibodies in CSF and serum, anddistribution/subcellular location of rhHNS in spinal cord, brain, andliver. In the recovery sacrifice monkeys, rhHNS effects were eitherabsent or significantly reduced. Thus, theno-observed-adverse-effect-level for the 6-month juvenile monkey studywas 8.3 mg/dose, the highest dose administered.

Monitoring changes in CSF cellularity and protein concentrations appearsto be a reliable correlate of the morphological changes noted onhistopathologic evaluation and may be useful in patients treated IT withrhHNS; these changes were considered to be an expected reaction to anIT-administered protein and were largely resolved after the recoveryperiod. These data from animal models provide confidence for pursuing ITtherapy as a treatment strategy for the neurological manifestations oflysosomal storage diseases. This juvenile nonhuman primate toxicologystudy demonstrates the feasibility and tolerability of administeringrhHNS via an IT lumbar drug delivery device to pediatric patients. Thenonadverse CNS pathology and lack of adverse clinical signs havesupported the recent investigational medical product dossier approvaland indicated that IT-administered rhHNS can safely and effectivelytreat CNS symptoms of Sanfillippo A syndrome.

Exemplary materials and methods used in various experiments described inthis example are provided below.

Study Design and rhHNS Dosing

The monkeys were randomized into five treatment groups; group 1 wasuntreated (implant device control [DC], port and catheter) and was notdosed with the vehicle or test article. Groups 2 through 5 received 0.6mL of 0, 2.5, 7.5 or 13.8 mg/mL rhHNS IT, (i.e., a total dose of 0, 1.5,4.5, or 8.3 mg) EOW. Four monkeys/sex/group were necropsied at 3 months(interim necropsy; 24 hours after the 6^(th) dose), fourmonkeys/sex/group (except the DC group, which were necropsied at 3months) were necropsied at 6 months of dosing (main necropsy; 24 hoursafter the 12^(th) dose), and the remaining four monkeys/sex/group werenecropsied at the end of a 1-month recovery period. At necropsy,selected tissues were harvested, processed, and examinedmicroscopically.

rhHNS was provided in an IT formulation vehicle consisting of 5 mMsodium phosphate, 145 mM sodium chloride, and 0.005% polysorbate 20 (pH7.0). Every other week doses of rhHNS were administered as a short-terminfusion over approximately eleven minutes: 0.6 mL (4 minutes) followedwith a flush of 0.5 mL phosphate-buffered saline (PBS) (7 minutes).Monkeys in the vehicle-control group received the IT formulation alone;DC monkeys received PBS (pH 7.2) IT.

Clinical Evaluation

Clinical signs and morbidity and mortality observations were recorded atleast twice daily starting at the first dose. Body weights were measuredprior to surgery, on the day of surgery, weekly during the study, and atnecropsy. Food consumption was monitored daily starting before surgery.Physical (heart rate, respiration, body temperature, auscultation, gait,disposition, abdominal palpation, lymph nodes, and general appearance)and neurologic (level of consciousness, tracking) examinations wereperformed before the study was initiated, each month during the study,and before necropsy. Motor functions, cerebral reflexes (pupillary,blink, and corneal reflex), and spinal reflexes (sensory foot, kneejerk, cutaneous, proprioceptive, and tail reflex) were also assessed.Electrocardiographic (ECG; leads I, II, and III) and ophthalmologicexaminations were completed prior to the first dose of rhHNS and in theweek before the interim (3-month) or the main (6-month) necropsy.Ophthalmic examinations were performed by indirect ophthalmoscope, themonkeys were sedated with ketamine HCl (IM, 8 mg/kg), and eyes weredilated with 1% tropicamide.

Clinical Pathology

Blood samples were collected from fasted monkeys for hematology andserum chemistry prior to the study start, after IT doses 1, 3, 5, 7, 9and 11, mid-recovery, and at necropsy. Urine samples were collected viapan catch predose, once monthly during the dosing and recovery period,and prior to necropsy. CSF samples were collected via the lumbarcatheter for total cell count and chemistry analysis at the time ofsurgery, and 24 hours following IT doses 1, 3, 5, 7, 9, 11,mid-recovery, and at necropsy; on occasion, samples were not collecteddue to partial catheter obstruction. Because higher than expected CSFleukocyte counts were noted, the 3-month dose 5 CSF samples werecollected from half the monkeys in each group before dosing and from theremaining monkeys 24 hours after dosing. The predose sample collectionoccurred at least 1 day prior to dosing so as not to significantly alterthe CSF volume just prior to dosing. For the 6-month and recoverymonkeys, CSF for total cell count and chemistry was collected from halfthe monkeys in each group before dosing and from the remaining monkeys24 hours after dosing. If a monkey had a nonsampling catheter due to anobstruction, a spinal tap (cisterna magna) was performed at thenecropsy.

rhHNS Analysis

Blood samples for rhHNS analysis were collected from a peripheral veinprior to and 24 hours post IT doses 2, 4, 6, 8, 10, 12; mid-recovery,and at necropsy. CSF samples were collected via the lumbar catheterprior to and 24 hours post IT doses 2, 4, 6, 8, 10, 12, mid-recovery,and at necropsy. rhHNS concentrations were determined by enzyme-linkedimmunosorbent assay. The capture antibody was a polyclonal rabbitanti-HNS IgG and the detection antibody was a horseradishperoxidase-conjugate of the same rabbit anti-HNS IgG. The LOD was 0.22ng/mL; thus, the LOQ was calculated to be 0.66 ng/mL. Serum and CSFsamples were screened in duplicate at 1:100 and 1:5 dilutions; samplesexceeding the high end of the calibration curve were further diluted andretested.

Anti-HNS Antibody Analysis

Blood for antibody analysis was collected from a peripheral veinapproximately 1 week prior to IT doses 2, 4, 6, 8, 10, 12; mid-recovery,and at necropsy. CSF samples for antibody analysis were collected atsurgery, and via the lumbar catheter approximately 1 week prior to ITdoses 2, 4, 6, 8, 10, 12; mid-recovery; and at necropsy. A Meso ScaleDiscovery (MSD®) technology electrochemiluminescent bridge test was usedfor detection of anti-HNS antibodies. The assay is a general, butsensitive, screening method for anti-HNS antibodies from any species andall immunoglobulin isotypes. The LOD was 5 ng/mL, and the samples werescreened in duplicate at a 1:20 dilution, resulting in an effectiveassay sensitivity of 100 ng/mL. Samples exceeding the high end of thecalibration curve were further diluted and retested.

Necropsy and Preparation of Tissues

Monkeys underwent a full necropsy either 24 hours after the final ITdose (main necropsy) or at the end of the 1-month recovery period(recovery necropsy). All monkeys were sedated with ketamine HCl (IM, 8mg/kg), were maintained on an isoflurane/oxygen mixture, and received anIV bolus of heparin sodium (200 IU/kg). Monkeys were perfused via theleft cardiac ventricle with room temperature 0.001% sodium nitrite insaline at a rate of 200 ml/min for 12 min (2400 ml). After collection,tissue samples were then fixed in 10% neutral buffered formalin forhistopathologic examination/immunohistochemical analysis or were frozenon dry ice and stored at −60° C. or lower for analysis of rhHNSactivity.

The brain was cut in a brain matrix (MBM-2000C, ASI Instruments, Inc.,Warren, Mich.) at 3-mm coronal slice thickness. The slices werenumbered, with the most rostral slice designated as slice 1. Slices 1,4, 7, 10, 13, and 16 were processed for histopathology and slices 2, 5,8, 11, 14, and 17 (if available) were processed forimmunohistochemistry. Slices 3, 6, 9, 12, and 15 were frozen foranalysis of rhHNS activity. The spinal cords (cervical, thoracic, andlumbar portions) were cut into 1-cm sections. The first slice and everythird slice thereafter were processed for histopathologic evaluation andthe second slice and every third slice thereafter were processed forimmunohistochemical analysis. The third slice and every third slicethereafter were frozen for rhHNS analysis. The distribution of sliceswas adjusted so that the slice containing the tip of the intrathecalcatheter (slice 0) was fixed in formalin and analyzed forhistopathology. Duplicate samples of ˜5 g of the liver were taken fromtwo separate lobes and frozen for rhHNS analysis and an additionalsample of ˜5 g was fixed for immunohistochemical analysis.

Histopathology

The brains, spinal cords, dorsal spinal nerve roots/ganglion, sciatic,tibial and sural nerves, a complete tissue list (typical for preclinicaldrug safety studies of this duration in this species), and any grosslesions were harvested at necropsy from all monkeys. Tissue sectionswere embedded in paraffin and stained with hematoxylin and eosin (inaddition to any special staining/embedding procedures noted below) forcomprehensive microscopic evaluation.

Brain sections from the prepared paraffin blocks from the device andvehicle-control groups, and the high-dose monkeys were stained withFluoro-Jade B (a stain increasing the sensitivity of evaluating neuronaldegeneration) and Bielschowsky's silver (a procedure that allows directvisualization of axons, dendrites, and neuronal filaments). TheFluoro-Jade B stained slides were examined under fluorescent lightingusing a fluorescein isothiocyanate filter cube.

Spinal cords were sectioned serially, with a transverse and obliquesections taken at the cervical, thoracic, and lumbar regions (one sliceexamined at each level) including sections at the catheter tip; anadditional transverse section was taken from the cauda equina region.Dorsal spinal roots and ganglia (midcervical, midthoracic, andmidlumbar) were processed and examined. Peripheral nerves (sciatic,tibial, and sural) were sectioned longitudinally, embedded in paraffinand stained with hematoxylin and eosin (H&E). Cross sections werepostfixed in osmium, embedded in Spurr's resin, sectioned (2 μm) andstained with toluidine blue. Serial spinal cord sections, as well asdorsal spinal nerve roots and ganglia, from the device and vehiclecontrol groups and the high-dose group were stained with Bielschowsky'ssilver. Spinal cord sections from these groups also were stained withanti-glial fibrillary acidic protein, an immunohistochemical stain thatallows for direct visualization of astrocytes and their processes.

Preparation of Tissue Extracts for Quantitative Analysis

Frozen brain slices 3, 6, 9, 12, and 15 were dissected by separating theleft and right hemispheres. Surface tissue was taken by measuring 4 mmfrom the surface, and the remaining tissue in each hemisphere wasconsidered deep tissue. If present (e.g., slices 6 and 9), an additionalperiventricular sample was cut from the coronal slices. Since onlyone-half of the brain (the right side) was processed (the left side wasretained frozen), the sectioning resulted in two to three samples perslice: right surface, right deep, and, if present, right periventricular(i.e., Ventricle deep; Vdeep). Cerebellar and brain stem tissues, whenpresent, were isolated prior to separating the hemispheres and wereprocessed independently. Spinal cord sections were prepared similarly,weighed, and homogenized.

Tissue samples were homogenized in lysis buffer (1 ml/0.25 g tissue)formulated with 10 mM Tris, 5 mM ethylenediaminetetracetic acid, 0.1%Igepal supplemented with Alpha Complete protease inhibitor minitablets(Roche Diagnostics, Indianapolis, Ind.) using TeenA Lysing Matrix Atubes or conical polypropylene tubes. Samples were processed for 40seconds in the Fastprep-24 automated homogenizer (MP Biomedicals, Solon,Ohio) or PowerGen Model 125 powered homogenizer (Omni International,Kennesaw, Ga.). Once homogenized, samples were subjected to fivefreeze-thaw cycles using an ethanol/dry ice bath and a 37° C. water bathand then centrifuged at 4° C. to pellet tissue debris; supernatants werestored at −80° C. until assayed. rhHNS activity was determined using aspecific substrate (4-methylumbelliferyl-α-D-N-sulphoglucosaminide) witha 2-step fluorometric assay.

Tissue Processing and Staining for Immunohistochemistry

Six formalin-fixed coronal brain slices (slice numbers 2, 5, 8, 11, 14,and 17) of 3-mm thickness from each monkey were numbered 1 to 6 rostralto caudal. Generally, slices 1 to 4 contained basalnuclei/thalamus/midbrain and cerebrum, and the caudal two slicescontained cerebellum and brain stem (medulla oblongata) tissue. Brain,spinal cord and liver sections (from the same paraffin blocks as thoseused for H&E and the various special stains) were immunohistochemicallystained for rhHNS. A specific mouse monoclonal antibody (clone 2C7;Maine Biotech, Portland, Me.) was used to detect intracellular uptake ofIT-administered rhHNS; this reagent demonstrated no cross-reactivitywith endogenous cynomolgus monkey rhHNS. Negative controls wereperformed using an irrelevant mouse IgG. Deparaffinized slides wereincubated with primary mouse anti-HNS antibody overnight at 2 to 8° C. Asecondary goat anti-mouse biotinylated immunoglobulin G was added andincubated for 30 minutes at 37° C. Avidin/biotinylated horseradishperoxidase complex was added and incubated for 30 minutes. Slides wereincubated in peroxidase substrate diaminobenzidine solution until thedesired stain intensity developed. Nuclei were counterstained withhematoxylin.

Statistical Analyses

Body weights, body weight changes, food consumption, respiratory rate,body temperature, heart rate, CSF cell count, CSF chemistry, clinicalpathology data, urine data, and absolute and relative organ weights wereanalyzed by a one-way analysis of variance and a comparison of thedevice and vehicle control groups to each rhHNS-dosed group by Dunnett'stest. In addition, the statistical analysis compared the two controlgroups to each other. Analysis was two-tailed for significance levels of5% and 1%. All data are presented as mean±standard deviation.

Example 5. Heparan N-Sulfatase Biodistribution and PharmacokineticStudies

The experiments in this example were designed to determine tissuedistribution of rhHNS in rats after a single intravenous or intrathecaldose (1 or 10 mg/kg) of rhHNS. For example, among other things, thepurpose of these experiments were to characterize the biodistribution(BD) properties of rhHNS in rats using positron emission tomography(PET); to compare distribution patterns of rhHNS when given in differentroutes (IV or IT) and at different doses (1 or 10 mg/kg); and todetermine pharmacokinetic properties of rhHNS in each of the interestorgans in these dosing regimens.

Pharmacokinetic (PK) and biodistribution (BD) profiles of¹²⁴I-sulfamidase (rhHNS) were studied by tissue PET imaging in ratsafter single intravenous (IV) or intrathecal (IT) administration of 1 or10 mg/kg of ¹²⁴I-HNS. Radioactivity-time data in the region of interestwere obtained from dynamic images in the first 20 min and from staticimages at 0.05 (only for IT administration), 1, 2, 4, 8, 24, 48, 96 and192 hours post IV or IT dosing.

Four rats in each of four groups (1 mg/kg IV, 1 mg/kg IT, 10 mg/kg IVand 10 mg/kg IT) were used in this study. Radioactivity-time data weremeasured in the head, brain (including cerebrospinal fluid, CSF), spineand liver regions after IT administration; and in the blood, brain(including CSF), liver, kidney, heart (including lungs) and skin afterIV administration. The data were corrected by the decay half-life of124-iodine (100.2 hours), expressed as percentage of injected dose (%ID) of a region of interest or % ID per gram (% ID/g) of the imagedtissues, and then normalized for the body weight of 200 grams. The totalamounts (ug) or concentrations (ug/g) of the dosed protein in the regionof interest were calculated from the corresponding % ID or % ID/g data.

In the first 20 min after IT dosing, total amount of rhHNS in the headregion was reduced at a constant rate of 0.002/min-0.011/min (λz) at 1and 10 mg/kg. Clearance rates and distribution volumes were not used forpharmacokinetic comparisons between the two doses and the twoadministration routes in this report (see Results section for moreinformation). The constant rates of elimination from the brain wereessentially the same at two test doses (λz: 0.016/hr versus 0.014/hr for1 and 10 mg/kg, respectively) with a similar half-life of about two daysas determined by static imaging up to 192 hours after IT dosing. Thevalues of Cmax and AUC (0-last or 0-infinite) were proportional to theadministered doses. A linear PK behavior was indicated in the dose rangeof 1 to 10 mg/kg given in these IT single-dosing regimens. Concentrationgradients were observed from the proximal to distal sections of thespine at both dose levels.

After IT dosing, rhHNS protein was measurable in the liver up to 96hours at 1 mg/kg and up to 192 hours at 10 mg/kg of rhHNS. Theconcentrations in the liver reached the peak 2 hours at 1 mg/kg, and 7hours at 10 mg/kg. The elimination was 0.030±0.011/hr (mean λz) at 1mg/kg, which was not significantly different from that at 10 mg/kg (λz0.017±0/hr) (p=0.10), with a corresponding t1/2 (28 versus 42 hours atthe doses of 1 and 10 mg/kg, respectively).

After IV dosing, the elimination half-lives in the liver, kidney, heartand skin were 47±10 and 38±13 hours for the liver, 54±25 and 29±16 hoursfor the kidney, 36±15 and 42±19 hours for the heart, and 40±21 and 31±13hours for the skin at 1 and 10 mg/kg, respectively; while the half-livesin the brain were 71±23 and 60±53 hours. The mean values of Cmax for theliver, skin, kidney, heart and brain were 9.6, 0.30, 0.25, 0.22, and0.08 ug/g at 1 mg/kg and 132, 7.9, 3.9, 3.7 and 1.8 ug/g at 10 mg/kg.After the Cmax values from individual animal were normalized for dose,the Cmax/dose values at 10 mg/kg were significantly higher than that at1 mg/kg in all these organs (most p values <0.05, p=0.06 for the liver).The values of AUClast for the liver, skin, kidney, heart and brain were525, 16, 14, 9 and 7 hr·ug/g at 1 mg/kg; and 6747, 276, 183, 201 and 86hr·ug/g at 10 mg/kg. After normalization, the AUClast/dose values at 10mg/kg were significantly higher than that at 1 mg/kg in the skin(p<0.01), marginally different in the heart (p=0.06), and notsignificantly different in the liver, brain and kidney (all p values>0.34).

When the same dose of rhHNS was injected, intrathecal administrationresulted in a three-log greater brain exposure than that withintravenous administration. The elimination half-life in the brain was 2days by IT and 3 days by IV administration. However, hepatic exposuresafter IT dosing were similar to that after IV dosing at the same dose ofrhHNS. The exposure (Cmax and AUClast) for the liver by IT/IV at 1 mg/kgand 10 mg/kg were in a range of 0.4-1.2.

Experimental Design

The central nervous system (CNS) is vulnerable in most lysosome storagediseases and is seriously damaged in some types of these diseases, suchas Sanfilippo (mucopolysaccharidosis III), Metachromatic Leukodystrophy(MLD) and Hunter Syndrome. As described herein, it is contemplated that,due to poor penetration through blood-brain barrier when administeredperipherally, direct administration of enzymatic proteins into the CNSmay increase their concentrations in the central nervous tissues andfurther enhance their therapeutic effects. Intrathecal (IT, or cisternamagna) administration was investigated and compared with IVadministration at different dose levels in this study.

PET is a non-invasive, repeatable and quantitative technology to providedynamic change of drug concentration over time in the organ of interest.The dynamic concentration-time data from target organs (active sites,rather than in blood circulation) are valuable, and are directly relatedto the biological activity of the dosed drug. Furthermore, theinformation on tissue exposures from PET study in animals can be used toguide the selection of the first-dose in human.

Materials and Methods Test Articles

Heparin N-Sulfatase (rhHNS) was formulated at a concentration of 20mg/mL of rhHNS in 5 mM sodium phosphates buffer with 145 mM sodiumchloride at pH 7.0. The material was purified by RP-HPLC and contained98.7% of Heparin N-Sulfatase with 99.9% of dimer. rhHNS was labeled with¹²⁴iodine.

Sample Source

Radioactivity images were from rats after IV and IT dosing¹²⁴I—H—N-sulfatase at 1 and 10 mg/kg.

Animals

Sixteen male Sprague-Dawley rats were purchased from Charles RiverLaboratories (190±60 g, n=16), and were separated into four groups(n=4). Single IV or IT injection at two different doses (1 mg/kg and 10mg/kg) was given to each group of these rats (total 4 groups). The doseand injected volume were individualized based on the body weight of eachanimal. In two IV-treated groups, sedation was induced by IV injectionof sodium pentobarbital at a dose of 35 mg/kg. Intravenous doses wereinjected in a bolus through a tail vein. In two IT-treated groups,animals were anesthetized by intra-peritoneal administration of sodiumpentobarbital at a dose of 50 mg/kg. Intrathecal doses were administeredover 1 min at cisterna magna level through the atlanto-occipitalmembrane. The actual administered radioactivity was measured by PET, andserved as the injected dose.

Experimental and/or Assay Method(s)

Dynamic images (every 2 min) were obtained in the first 20 minutes inthe regions of the heart (including the lungs), liver and kidneys postIV injection; and in the head region post IT administration of bothdoses. Static imaging was acquired in the regions including the brain(including cerebrospinal fluid, CSF), liver, kidney, heart (includingthe lungs), muscle, skin and bone in IV-treated group; and in the regionof head, brain (including CSF) and liver of IT-treated animals at 0.05(only available for IT groups), 1, 2, 4, 8, 24, 48, 96 and 192 hourspost-dosing. The images were reconstructed and the three body sectionswere fused into one image.

Data Analyses

PET data were expressed in nanocurie (nCi) per mL (for fluid) or pergram (for tissue). Relative activity was obtained for the brain, liver,kidneys, skeletal muscle, stomach, heart (with lungs) and skin regionsin static images. Absolute activity in the whole head or brain regionswas obtained for the animals that received IT injections. Radioactivityper millimeter of spinal column was determined in the IT injectedanimals at three selected sections: the proximal (neck), mid (againstupper edge of the liver), and distal (1 cm from the distal end of theprotein containing compartment) spine.

All data were corrected by the decay half-life of ¹²⁴I (100.2 hours) andnormalized for registration efficacy based on calibration with a ¹²⁴Isource with externally measured activity. The data were then expressedas percentage of injected dose (% ID) of a whole region (the head andbrain) or % ID per gram (% ID/g) of a tissue, and then normalized for abody weight of 200 grams [data normalization: (% ID or % ID/g)/bodyweight of the animal×200]. The normalization was adopted to reduce thevariability of the data, as only four animals were used in each group.

In this example, rhHNS protein concentrations or amount were calculatedusing the injected protein dose to each animal: protein concentration(ug/g)=(% ID/g)×(mg/kg of injected dose×1000×0.2); total amount of thedosed protein (ug) in a region of interest=% ID×(mg/kg of injecteddose×1000×0.2), here the injected dose was 1 mg/kg or 10 mg/kg and 0.2is the normalizing factor for body weight. Group mean and standarddeviation of each PK parameter were calculated based on the individualnon-compartmental data in each of the four groups. A Student t-test wasperformed to compare the values of λz, t1/2, Cmax and AUC between thetwo test doses and the two administration routes. Statisticalsignificance was defined as a p-values less that 0.05 (p<0.05).

Results

The amounts (ug) or concentrations (ug/g) of rhHNS in the followingtables, figures and PK analyses were calculated by multiplying theinjected protein dose (1 mg/kg or 10 mg/kg) with the correspondingvalues of % ID or % ID/g.

Intrathecal Treatment with ¹²⁴I-HNS at Doses of 1 and 10 mg/kg

The amount of the dosed protein (ug) in the head region from dynamicimages was plotted as a function of time in FIG. 17. The concentration(ug/g) in the brain regions from static images was plotted as a functionof time in FIG. 18. The total amount of injected protein (ug) in thebrain and head regions from static images were plotted with time in FIG.19 and FIG. 20 respectively. Concentration-time curves (ug/mm) at theproximal, mid and distal spine were shown in FIG. 21 to FIG. 23. FIG. 24shows the changes of rhHNS concentration (ug/g) in the liver with timeafter IT administration of ¹²⁴I-HNS at 1 and 10 mg/kg.

The total amount-time (ug) or concentration-time (ug/g) data wereanalyzed by non-compartmental models (WinNonlin 5.2, Pharsight, MountainView, Calif.). The PK parameters, such as the constant rate ofelimination (λz), peak concentration (Cmax), terminal half-life (t1/2),area under curve (AUClast and AUCO-inf) and others were estimated fromthe data of each individual animal.

Clearance rates and distribution volumes were estimated (see Appendix3), however, they were not used for PK comparisons between the two dosesand the two administration routes in this report for two reasons (1)this study focused on biodistribution of rhHNS in solid tissues, ratherthan on blood PK; and (2) the radioactivity in the brain region was thesum of those from the brain tissue (solid) and CSF (liquid), which couldnot be separated from each other in the study. The λz was evaluated, andused for comparison, because it indicated a percentage of the injecteddose eliminated per unit of time.

The group means and standard deviations (SD) were calculated andcompared between two test doses. These PK parameters are tabulated inTable 23 below:

TABLE 23 Summary of non-compartmental PK parameters (group mean ± SD) invarious organs after IT and IV dosing 1 and 10 mg/kg of ¹²⁴I-HNS. Brain(ug/g)* Liver Brain (ug)^(#) Head (ug)^(#) Parameter Mean SD Mean SDMean SD Mean 1 mg/kg IT λz 0.016 0.003 0.030 0.011 0.017 0.002 0.016t_(1/2) 45 7 28 16 42 5 45 T_(max) 0.1 0.0 2.3 1.3 2.0 4.0 0.1 C_(max)257.0 89.9 4.9 1.3 68.6 8.0 200.1 AUC_(last) 8393 2457 204 50 3809 6228216 AUC_(inf.) 8942 2416 216 57 4030 643 8904 MRT_(last) 46 6 32 13 445 46 10 mg/kg IT λz 0.014 0.001 0.017 0.000 0.014 0.001 0.010 t_(1/2) 494 42 1 51 5 70 T_(max) 0.1 0.0 7.0 2.0 0.1 0.0 0.1 C_(max) 2628 265 10541 836 117 1844 AUC_(last) 83962 10083 7987 3276 59115 8624 128751AUC_(inf.) 89460 12098 8345 3424 63836 9466 151405 MRT_(last) 56 1 51 158 2 65 Head (ug)^(#) Proximal Mid Distal Parameter SD Mean SD Mean SDMean SD 1 mg/kg IT λz 0.002 0.025 0.012 0.020 0.008 0.028 0.016 t_(1/2)7 32 13 39 16 30 12 T_(max) 0.0 0.3 0.5 1.8 1.5 1.0 0.0 C_(max) 0.0 0.50.1 0.2 0.0 0.1 0.0 AUC_(last) 782 9 3 7 3 2 1 AUC_(inf.) 1069 11 3 8 33 2 MRT_(last) 5 31 17 34 20 16 5 10 mg/kg IT λz 0.001 0.018 0.008 0.014— 0.006 — t_(1/2) 9 45 18 50 — 123 — T_(max) 0.0 0.3 0.5 8.7 13.3 8.0 —C_(max) 314 6 4 1 0 1 — AUC_(last) 15723 83 67 35 20 38 — AUC_(inf.)15123 98 66 60 — 73 — MRT_(last) 3 31 2 32 7 61 — Brain (ug/g)* LiverKidney Heart Skin 1 mg/kg IV λz 0.011 0.005 0.015 0.003 0.016 0.0090.021 0.006 0.021 0.010 t_(1/2) 71 23 47 10 54 25 36 15 40 21 T_(max) 712 5 4 10 12 2 1 5 4 C_(max) 0.1 0.0 9.6 1.5 0.2 0.1 0.2 0.0 0.3 0.1AUC_(last) 7 2 525 104 14 5 9 3 16 4 AUC_(inf.) 9 3 576 138 16 6 10 3 185 MRT_(last) 61 16 47 5 47 18 36 13 41 16 10 mg/kg IV λz 0.102 0.1800.021 0.012 0.035 0.024 0.020 0.010 0.026 0.012 t_(1/2) 60.5 53.1 37.813.4 28.4 16.4 41.6 18.6 31.0 12.7 T_(max) 13 12 2 1 12 11 16 9 3 1C_(max) 1.8 0.2 131.6 26.8 3.9 0.7 3.7 0.7 7.9 2.3 AUC_(last) 86 66 67472837 183 123 201 89 276 40 AUC_(inf.) 118 98 7171 3029 198 131 230 110292 43 MRT_(last) 43 32 40 14 33 21 41 18 33 13

In the first 20 min after dosing, total amount (ug) of rhHNS in the headregion was reduced at a constant rate of 0.002-0.011 per min (λz,0.005±0.004/min) at 1 mg/kg and 0.003-0.010 per min (0.007±0.003/min) at10 mg/kg. These constant rates of elimination were not significantlydifferent at these two dose levels (p=0.57, FIG. 17).

The concentration-time curve (ug/g from 0.05 to 192 hours) for the brainindicated a bi-phasic profile (FIG. 18). The early phase lasts for abouttwo hours. The terminal phase follows first-order kinetics. The constantrates of elimination from the brain were very similar at two testeddoses (0.0016±0.003 and 0.014±0.001 per hour) with a similar half-lifeof about two days (45±7 and 49±4 hours at 1 and 10 mg/kg, respectively).The values of peak concentrations (257±90 and 2628±265 ug/g) and AUClast(8393±2457 and 83962±10083 hr·ug/g at 1 and 10 mg/kg, respectively)increase approximately ten-fold when the dose was increased from 1 to 10mg/kg. These observations indicated a linear PK behavior in the doserange of 1 to 10 mg/kg given in these IT single dosing regimens. Thepeak concentration appeared in the brain 3 min (Tmax) after IT dosing.

The total amount-time curve (ug from 0.05 to 192 hours) in the brain andhead regions followed the same bi-phasic pattern as seen withconcentration-time curves (ug/g) in the brain (FIG. 19 and FIG. 20). Thevalues of Cmax in the brain region were significantly lower than that inthe head region (69±8 versus 200±0 at 1 mg/kg, p<0.01; and 836±117versus 1844±314 ug, p<0.01 at 10 mg/kg, respectively). The constantrates of elimination were 0.017±0.002/hr and 0.014±0.001/hr for thebrain, and 0.016±0.002 and 0.010±0.001/hr for the head region at 1 and10 mg/kg, respectively. The values of mean residual time were 42±5versus 51±5 hours for the brain (p=0.048), and 45±7 versus 70±9 hoursfor the head (p<0.01) at 1 and 10 mg/kg, respectively. Theseobservations suggested that the dosed protein was eliminated from bothregions more rapidly at lower dose than at higher doses. The meanhalf-lives were in a range of 42 to 70 hours in these regions after ITdosing 1 mg/kg and 10 mg/kg of rhHNS.

A concentration gradient was observed from the proximal, to the mid andto the distal sections of the spine at both dose levels (data notshown). After IT dosing, the peak concentration (ug/mm of spine column)was seen around 30 min (0 to 1 hour) at the proximal, 1 to 4 hours atthe mid (except of one rat being 24 hours) and 1 to 8 hours at thedistal section. The half-lives in these sections were variable (meant1/2: 32±13 and 45±18 hours for the proximal, 39±16 and about 50 hoursfor the mid, and 30±12 and about 123 hours for the distal sections ofspine at 1 mg/kg and 10 mg/kg, respectively). The mean values of peakconcentrations were roughly proportional to the doses at each of thesethree sections at 1 and 10 mg/kg of ¹²⁴I-HNS (0.5 versus 6.0, 0.2 versus0.9 and 0.1 versus 0.5 ug/mm at the proximal, mid and distal sections ofthe spine, respectively). The mean values of AUClast followed the sameproportional pattern as seen in the peak concentration (9.5 versus 83,6.8 versus 35, and 2 versus 38 hr·ug/mm at the proximal, mid and distalsections, respectively).

Even though rhHNS was not detectable in most peripheral organs, it wasmeasurable in the liver from as early as 1 hour (the first imaging timepoint after dosing) to 96 hours (three of four animals) at 1 mg/kg andto 192 hours (all four rats) at 10 mg/kg after IT dosing (FIG. 24). Theconcentrations in the liver reached the peak 2 hours after IT dosing of1 mg/kg, and 7 hours after IT dosing of 10 mg/kg, which was followed byan elimination phase with first-order kinetics. The constant rate ofelimination was faster at 1 mg/kg (λz 0.030±0.011/hr) than that at 10mg/kg (λz 0.017±0/hr) (p=0.10), with a corresponding shorter t1/2 (28±16versus 42±1 hours at the doses of 1 and 10 mg/kg, respectively, p=0.76).The value of AUClast at 1 mg/kg reduced about 40-fold in comparison withthat at 10 mg/kg (204±50 versus 7987±3276 ug/g, respectively).

Intravenous Treatment with ¹²⁴I-HNS at Doses of 1 and 10 mg/kg

The concentration in the brain, liver, kidney, heart (including lungtissue) and skin were plotted as a function of time after IV dosing 1and 10 mg/kg of rhHNS as shown in FIG. 25 through FIG. 29, respectively.Since the first static imaging time point for these organs was one hourafter dosing, the initial phase of these concentration-time curvescannot be observed in this study. The concentration-time curves for theliver, kidney, heart and skin showed a flat phase from 1 to 8 hoursafter IV dosing. This flat phase lasted for 24 hours in the brainpost-dosing, suggesting that the brain took up the IV dosed proteinslower than that by the peripheral organs. The remaining data indicateda terminal elimination phase with approximately first-order kinetics.

The elimination half-lives in the liver, kidney, heart and skin 47±10and 38±13 hours for the liver, 54±25 and 29±16 hours for the kidney,36±15 and 42±19 hours for the heart and 40±21 and 31±13 hours for theskin at 1 and 10 mg/kg, respectively; while the half-lives in the brainwere 71±23 and 60±53 hours (Rat 3 in 10 mg/kg group was excluded forinsufficient data to determine t1/2) at 1 and 10 mg/kg, respectively. Nostatistical differences were seen between the half-lives at 1 and 10mg/kg in these organs, with an exception of p value <0.03 for kidney.

The mean values of Cmax for the liver, skin, kidney, heart and brainwere 9.6, 0.3, 0.25, 0.22, and 0.08 ug/g at 1 mg/kg and 132, 7.9, 3.9,3.7 and 1.8 ug/g at 10 mg/kg. The ratios of Cmax at 10 mg/kg to thecorresponding values at 1 mg/kg were 14, 26, 16, 17 and 23 for theseorgans. After the Cmax values from individual animal were normalized fordose, the Cmax/dose values at 10 mg/kg were significantly higher thanthat at 1 mg/kg in all these organs (most p values <0.05, p=0.06 for theliver). The values of AUClast for the liver, skin, kidney, heart andbrain were 525, 16, 14, 9.3 and 7 hr·ug/g at 1 mg/kg; and 6747, 276,183, 201 and 86 hr·ug/g at 10 mg/kg. The ratios of AUClast at 10 mg/kgto the corresponding values of AUClast at 1 mg/kg were 13, 17, 13, 22and 12 for these organs, respectively. After normalization, theAUClast/dose values at 10 mg/kg were significantly higher than that at 1mg/kg in the skin (p<0.01), marginally different in the heart (p=0.06),and not significantly different in the liver, brain and kidney (all pvalues >0.34).

These observations suggested (1) the half-lives in most organs wereabout 2 days, with the exception of the brain (about 3 days); (2) theexposure per gram in the liver was larger than that of the skin, heartand kidney, which are larger than that of the brain; (3) with a ten-foldincrease in dose (10/1 mg/kg), the values of Cmax at 10 mg/kg from alltested organs increased more than 10 times than that at 1 mg/kg.

The peak concentration in the brain was reached 1-24 hours (Tmax) afterIV dosing.

Comparison of IV Versus IT Treatments

The concentration-time curves in the brain and liver after IV and ITadministration at 1 and 10 mg/kg are compared in FIG. 30 and FIG. 31,respectively. The ratios of Cmax in the brain by IT/IV at 1 and 10 mg/kgwere 3212 and 1501, respectively. These ratios of AUCO-192 hr were 1136and 978. These observations indicated that, when the same dose of rhHNSwas injected, intrathecal administration resulted in an approximatelythree-log greater exposure of the brain than that with intravenousadministration. The elimination half-life in the brain was 2 days (45and 49 hours at 1 and 10 mg/kg) by IT and 3 days (71 and 60 hours at 1and 10 mg/kg) by IV administration at both dose levels.

However, hepatic exposures after IT dosing were similar to that after IVdosing at the same dose of rhHNS. The ratios of Cmax in the liver byIT/IV at 1 mg/kg and 10 mg/kg were 0.5 and 0.8, and the ratios ofAUClast were 0.4 and 1.2, respectively.

Conclusions

Pharmacokinetic and biodistribution profiles of ¹²⁴I-sulfamidase (rhHNS)were studied by tissue PET images in rats after single intravenous orintrathecal administration of 1 or 10 mg/kg of ¹²⁴I-sulfamidase.Concentration-time data were obtained both dynamically (the first 20min) and statically in the regions of interest at 0.05, 1, 2, 4, 8, 24,48, 96 and 192 hours post dosing. By dynamic imaging after IT dosing,total amount of rhHNS in the head region was reduced at a similarconstant rate of 0.005/min-0.007/min (mean λz) in the first 20 min. Bystatic imaging, the rates of elimination from the brain were essentiallythe same at two tested doses (λz: 0.016/hr versus 0.014/hr for 1 and 10mg/kg, respectively) with a similar half-life about two days.

The values of Cmax and AUClast were proportional to the administereddoses, and a linear PK behavior was indicated in the dose range of 1 to10 mg/kg given in these IT single dosing regimens.

Concentration gradients were observed from the proximal to distal spineat both dose levels.

After IT dosing, the peak concentration was seen around 20 min at theproximal, 1 to 4 hours at the mid and 1 to 8 hours at the distalsections. Linear PK behavior was also indicated in the differentsections of the spine.

After IT dosing, rhHNS protein was measurable in the liver from veryearly time up to 96 hours at 1 mg/kg and 192 hours at 10 mg/kg. The rateof elimination was faster at 1 mg/kg (λz 0.030/hr) than that at 10 mg/kg(λz 0.017/hr), with a corresponding shorter t1/2 at the lower dose(28±16 versus 42±1 hours at the doses of 1 and 10 mg/kg, respectively).

After IV dosing, the elimination half-lives in the liver, kidney, heartand skin 47±10 and 38±13 hours for the liver, 54±25 and 29±16 hours forthe kidney, 36±15 and 42±19 hours for the heart and 40±21 and 31±13hours for the skin at 1 and 10 mg/kg, respectively; while the halflivesin the brain were 71±23 and 60±53 hours. The mean values of Cmax for theliver, skin, kidney, heart and brain were 9.6, 0.30, 0.25, 0.22, and0.08 ug/g at 1 mg/kg and 132, 7.9, 3.9, 3.7 and 1.8 ug/g at 10 mg/kg.After the Cmax values from individual animal were normalized for dose,the Cmax/dose values at 10 mg/kg were significantly higher than that at1 mg/kg in all these organs (most p values <0.05, p=0.06 for the liver).The values of AUClast for the liver, skin, kidney, heart and brain were525, 16, 14, 9.3 and 7 hr·ug/g at 1 mg/kg; and 6747, 276, 183, 201 and86 hr·ug/g at 10 mg/kg. After normalization, the AUClast/dose values at10 mg/kg were significantly higher than that at 1 mg/kg in the skin(p<0.01), marginally different in the heart (p=0.06), and notsignificantly different in the liver, brain and kidney (all p values>0.34).

Example 6. Treatment of Sanfilippo A (San A) Patients with rhHNS

Direct CNS administration through, e.g., IT delivery can be used toeffectively treat San A patients. This example illustrates a multicenterdose escalation study designed to evaluate the safety of up to 3 doselevels every other week (EOW) for a total of 40 weeks of rhHNSadministered via an intrathecal drug delivery device (IDDD) to patientswith San A. Various exemplary intrathecal drug delivery devices suitablefor human treatment are depicted in FIGS. 32-35.

In one particular example, up to 16 patients will be enrolled:

Cohort 1: 4 patients (Lowest Dose—10 mg)

Cohort 2: 4 patients (Intermediate Dose—30 mg)

Cohort 3: 4 patients (Highest Dose—100 mg)

4 patients will be randomized to no treatment or use of device.

Sanfilippo Syndrome Type A patients generally demonstrate cognitive andneurodevelopmental impairment including delay of early developmentmilestones (e.g., walking, speech, toilet training), intellectualdeficit, hyperactivity, hearing loss, impaired speech development,deficits in motor skills, hyperactivity, aggressiveness and/or sleepdisturbances, among others. All of the indications can be part of thecriteria for trials. Patients are selected for the study based oninclusion of the following criteria: (1) 3-18 years of age; (2)intelligence quotient of less than 77 or a decline of 15 to 30 IQ pointsin past 3 years; (3) no CSF shut or poorly controlled seizure disorderand (4) no co-morbidities presenting anesthesia and/or surgical risks.

Safety of ascending doses of rhHNS administered by IT injection for 6months in children with late infantile Sanfilippo Syndrome Type A isdetermined. Enrollment and escalation will be very slow to provide fullassessments of patient safety. In addition, the clinical activity ofrhHNS on gross motor function, and single and repeated-dosepharmacokinetics in serum and concentrations in cerebrospinal fluid(CSF) are assessed.

Objectives of the study will be to evaluate the safety and tolerabilityof ascending doses of rhHNS, as well as the safety, tolerability andlong term patency of the IDDD. Additionally, the concentration of rhHNSafter single and repeated IT doses in both CSF and blood, as well as theeffects of rhHNS on CF biomarkers and urinary GAG. Further evaluationwill include effects of rhHNS on clinical parameters such asphysiological and neurocognitive assessments, neuro function and brainstructure volumes. Additionally, the effects of treatment on dailyliving and relationships between biomarkers and symptoms can beevaluated.

Typically, treatment of Sanfilippo Syndrome Type A patients by ITdelivery of rhHNS results in reduction of accumulation of GAG in varioustissues (e.g., the nervous system, kidneys, gallbladder, and otherorgans).

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds of theinvention and are not intended to limit the same.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification. Thepublications, websites and other reference materials referenced hereinto describe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference.

1. A stable formulation for intrathecal administration comprising aheparan N-sulfatase (HNS) protein at a concentration at or greater than5 mg/ml, and no greater than 50 mM phosphate.
 2. The stable formulationof claim 1, wherein the HNS protein is present at a concentration up toapproximately 100 mg/ml.
 3. The stable formulation of claim 1, whereinthe HNS protein is present at a concentration up to approximately 30mg/ml.
 4. The stable formulation of claim 1, wherein the HNS protein ispresent at a concentration ranging from approximately 10-20 mg/ml. 5.The stable formulation of claim 1, wherein the HNS protein is present ata concentration ranging of approximately 15 mg/ml.
 6. The stableformulation of claim 1, wherein the HNS protein comprises an amino acidsequence of SEQ ID NO:1.
 7. The stable formulation of claim 1, whereinthe formulation further comprises NaCl.
 8. The stable formulation ofclaim 7, wherein the NaCl is present at a concentration ranging fromapproximately 0-300 mM.
 9. The stable formulation of claim 8, whereinthe NaCl is present at a concentration of approximately 100-200 mM. 10.The stable formulation of claim 9, wherein the NaCl is present at aconcentration of approximately 145 mM.
 11. The stable formulation ofclaim 1, wherein the formulation further comprises a polysorbatesurfactant.
 12. The stable formulation of claim 11, wherein thepolysorbate surfactant is polysorbate
 20. 13-17. (canceled)
 18. Thestable formulation of claim 1, wherein the phosphate is present at aconcentration no greater than 20 mM.
 19. The stable formulation of claim1, wherein the phosphate is present at a concentration of approximately5 mM.
 20. The stable formulation of claim 1, wherein the formulation hasa pH of approximately 5.0-8.0. 21-43. (canceled)
 44. A method oftreating Sanfilippo A Syndrome comprising a step of administeringintrathecally to a subject in need of treatment a formulation comprisinga heparan N-sulfatase (HNS) protein at a concentration at or greaterthan 5 mg/ml, and no greater than 50 mM phosphate. 45-53. (canceled) 54.The method of claim 44, wherein the intrathecal administration of theformulation results in reduction of lysosomal storage in the braintarget tissues, spinal cord neurons and peripheral target tissues.55-62. (canceled)
 63. The method of claim 44, wherein the intrathecaladministration of the formulation results in reduced intensity,severity, or frequency, or delayed onset of at least one symptom orfeature of the Sanfilippo A Syndrome. 64-67. (canceled)
 68. The methodof claim 44, wherein the intrathecal administration is used inconjunction with intravenous administration. 69-71. (canceled)
 72. Themethod of claim 44, wherein the intrathecal administration is used inabsence of concurrent immunosuppressive therapy.